Soft Protease Inhibitors and Pro-Soft Forms Thereof

ABSTRACT

The invention provides compounds and methods for inhibiting proteases. One aspect of the invention features pro-soft inhibitors which react with an activating protease to release an active inhibitor moiety in proximity to a target protease. In certain instances, compounds inhibit proteasomes and/or post-proline cleaving enzymes (PPCE), such as dipeptidyl peptidase IV. The compounds of the invention provide a better therapeutic index, owing in part to reduced toxicity and/or improved specificity for the targeted protease.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/752,017, filed Dec. 19, 2005.

BACKGROUND OF THE INVENTION

Proteases are enzymes that cleave proteins at specific peptide bonds.Proteases can be classified into four generic classes: serine, thiol orcysteinyl, acid or aspartyl, and metalloproteases (Cuypers et al., J.Biol. Chem. 1982, 257, 7086. Proteases are essential to a variety ofbiological activities, such as digestion, formation and dissolution ofblood clots, reproduction, and immune reaction to foreign cells andorganisms. However, aberrant proteolysis is associated with a number ofdiseases in humans and other mammals. Accordingly, it is oftenbeneficial to disrupt the function of one or more proteolytic enzymes inthe course of treating a patient.

The binding site for a peptide substrate consists of a series of“specificity subsites” across the surface of the enzyme. The term“specificity subsite” refers to a pocket or other site on the enzymecapable of interacting with a portion of a substrate for the enzyme. Indiscussing the interactions of peptides with proteases, e.g., serine andcysteine proteinases, the present application utilizes the nomenclatureof Schechter and Berger (Biochem. Biophys. Res. Commun. 1967, 27,157-162). The individual amino acid residues of a substrate or inhibitorare designated P1, P2, etc. and the corresponding subsites of the enzymeare designated 51, S2, etc., starting with the carboxy terminal residueproduced in the cleavage reaction. The scissile bond of the substrate isthe amide bond between P1-P1′ of the substrate. Thus, for a peptideXaa1-Xaa2-Xaa3-Xaa4, which is cleaved between the Xaa3 and Xaa4residues, the Xaa3 residue is referred to as the P1 residue and binds tothe S1 subsite of the enzyme, Xaa2 is referred to as the P2 residue andbinds to the S2 subsite, and so forth.

Dipeptidyl peptidase IV (DPIV or DPPIV) is a serine protease thatcleaves N-terminal dipeptides from a peptide chain containing,preferably, a proline residue in the penultimate position, e.g., in theP1 position. DPIV belongs to a group of cell-membrane-associatedpeptidases and, like the majority of cell-surface peptidases, is a typeII integral membrane protein, being anchored to the plasma membrane byits signal sequence. DPIV is found in a variety of differentiatedmammalian epithelia, endothelia and hematopoetic cells and tissues,including those of lymphoid origin where it is found specifically on thesurface of CD4⁺ T cells. DPIV has been identified as the leukocytedifferentiation marker CD26.

Proteasomes are cellular complexes comprising proteases responsible forthe majority of intracellular protein turnover in eukaryotic cells,including proteolytic degradation of damaged, oxidized or misfoldedproteins, as well as processing or degradation of key regulatoryproteins required for various cellular functions, such as cell cycleprogression. For example, the 26S proteasome is a multi-catalyticprotease comprising at its catalytic core the 20S proteasome, amulti-subunit complex of approximately 700 kDa molecular weight. Whileserving an essential physiological role, the proteasome is alsoresponsible for the inappropriate or accelerated protein degradationthat occurs as a result or cause of pathological conditions in whichnormal cellular processes become disregulated. One notable example iscancer, in which the unregulated proteasome-mediated degradation of cellcycle regulatory proteins, including cyclins, cyclin dependent kinaseinhibitors, and tumor suppressor genes, results in accelerated anduncontrolled mitosis, thereby promoting cancer growth and spread.(Goldberg et al. Chem.& Biol. 1995, 2, 503-508; Coux et al. Ann. Rev.Biochem., 1996, 65, 801-847; Deshaies, Trends Cell Biol. 1995, 5,428-434). Inhibition of proteasome enzymatic function holds promise inarresting or blunting disease progression in disease states such ascancer or inflammation.

Proteasome inhibitors, e.g., lactacystin and its analogs, have beenshown to block the development of the preerythrocytic and erythrocyticstages of Plasmodium spp, the malaria parasites. During both its hepaticand erythrocytic stages, the parasite undergoes radical morphologicalchanges and many rounds of replication, events that likely requireproteasome activity. Lactacystin has been found to covalently modify thecatalytic N-terminal threonines of the active sites of proteasomes,inhibiting the activity of all proteasomes examined, including those inmammalian cells, protozoa, and archeae. (Gantt et al. Antimicrob. AgentsChemother. 1998, 42, 2731-2738).

The human fibroblast activation protein (FAPα) is a M_(r) 95,000 cellsurface molecule originally identified with monoclonal antibody (mAb)F19 (Rettig et al. Proc. Natl. Acad. Sci. USA 1988, 85, 3110-3114;Rettig et al. Cancer Res. 1993, 53, 3327-3335). The FAPα cDNA codes fora type II integral membrane protein with a large extracellular domain,trans-membrane segment, and short cytoplasmic tail (Scanlan et al. Proc.Natl. Acad. Sci. USA 1994, 91, 5657-5661; WO 97/34927). FAPα shows 48%amino acid sequence identity to the T-cell activation antigen CD26, alsoknown as dipeptidyl peptidase IV (DPP IV), a membrane-bound protein withdipeptidyl peptidase activity (Scanlan et al.). FAPα has enzymaticactivity and is a member of the serine protease family, with serine 624being critical for enzymatic function (WO 97/34927). Work using amembrane overlay assay revealed that FAPα dimers are able to cleaveAla-Pro-7-amino-4-trifluoromethyl coumarin,Gly-Pro-7-amino-4-trifluoromethyl coumarin, andLys-Pro-7-amino-4-trifluoromethyl coumarin dipeptides (WO 97/34927).

FAPα is selectively expressed in reactive stromal fibroblasts of manyhistological types of human epithelial cancers, granulation tissue ofhealing wounds, and malignant cells of certain bone and soft tissuesarcomas. Normal adult tissues are generally devoid of detectable FAPα,but some foetal mesenchymal tissues transiently express the molecule. Incontrast, most of the common types of epithelial cancers, including >90%of breast, non-small-cell lung, and colorectal carcinomas, containFAPα-reactive stromal fibroblasts (Scanlan et al.). These FAPα⁺fibroblasts accompany newly-formed tumor blood vessels, forming adistinct cellular compartment interposed between the tumor capillaryendothelium and the basal aspect of malignant epithelial cell clusters(Welt et al. J. Clin. Oncol. 1994, 12, 1193-1203). While FAPα⁺ stromalfibroblasts are found in both primary and metastatic carcinomas, thebenign and premalignant epithelial lesions tested (Welt et al.), such asfibroadenomas of the breast and colorectal adenomas, only rarely containFAPα⁺ stromal cells. Based on the restricted distribution pattern ofFAPα in normal tissues and its uniform expression in the supportingstroma of many malignant tumors, clinical trials with ¹³¹I-labeled mAbF19 have been initiated in patients with metastatic colon carcinomas(Welt et al.).

SUMMARY OF THE INVENTION

One aspect of the present invention features compounds which inhibit aprotease. In certain instances, the compound is a pro-soft inhibitor.The pro-soft inhibitor is an inactive agent that is activated, i.e.,cleaved by an “activating protease,” to release an active inhibitormoiety in proximity to a “target protease.” The identity of theactivating protease and target protease can be the same or different.After activation of the pro-soft inhibitor, the active inhibitor moietyundergoes self-inactivation by proto-deboronation. Another aspect of thepresent invention is that the irreversible proto-deboronation stepproduces innocuous boric acid, which is expected to yield an improvedsafety profile (fewer side effects).

The invention features inhibitors for a wide array of proteases. Forexample, the inhibitor of the invention may inhibit post-prolinecleaving enzymes (PPCE), such as dipeptidyl peptidase IV. In certaininstances, the inhibitor of the invention inhibits proteasome activity.In other instances, the invention provides a pro-soft inhibitor that isactivated by a fibroblast activation protein to release a compound thatinhibits prostrate specific antigen (PSA). In still other instances, theinvention provides a pro-soft inhibitor that is activated by a prostratespecific antigen (PSA) to release a compound that inhibits proteasomeactivity. In a certain embodiment, the present invention providespro-soft inhibitors which inhibit post-proline cleaving enzymes, such asdipeptidyl peptidase IV.

Certain compounds of the invention have extended duration. Accordingly,in certain embodiments, the inhibitor is selected, and the amount ofinhibitor formulated, to provide a dosage which inhibits serum DPP IVlevels by at least 50% for at least 4 hours after a single dose, andeven more preferably for at least 8 hours or even 12 or 16 hours after asingle dose. For instance, in certain embodiments, the dosage isselected in an amount effective to improve one or more aberrant indicesassociated with glucose metabolism disorders (e.g., glucose intolerance,insulin resistance, hyperglycemia, hyperinsulinemia and Type I and IIdiabetes) over a 24 hour period.

Another aspect of the invention relates to a method of treatingdisorders and conditions by administering a protease inhibitor of theinvention. In certain instances, the disorder is one that is mediated byDPP IV. In certain instances, the subject inhibitors can be used toup-regulate GIP and GLP-1 activities, e.g., by increasing the half-lifeof those hormones or as part of a treatment for regulating glucoselevels and/or metabolism. In certain instances, the inhibitors can beused to reduce insulin resistance, treat hyperglycemia,hyperinsulinemia, obesity, hyperlipidemia, hyperlipoprotein-emia (suchas chylomicrons, VLDL and LDL), and/or regulate body fat and moregenerally lipid stores. In certain instances, the inhibitors of theinvention may be used to treat metabolism disorders, such as thoseassociated with diabetes, obesity and atherosclerosis. While not wishingto be bound by any particular theory, it is observed that compoundswhich inhibit DPP IV are able to improve glucose tolerance throughmechanisms involving DPP IV inhibition.

In other embodiments, the invention features a method of administering aDPP IV pro-soft inhibitor in an amount effective to improve aberrantindices associated with obesity. Fat cells release the hormone leptin,which travels in the bloodstream to the brain and, through leptinreceptors there, stimulates production of GLP-1. GLP-1, in turn,produces the sensation of being full. The leading theory is that the fatcells of most obese people probably produce enough leptin, but leptinmay not be able to properly engage the leptin receptors in the brain,and so does not stimulate production of GLP-1. There is accordingly agreat deal of research towards utilizing preparations of GLP-1 as anappetite suppressant. The subject method provides a means for increasingthe half-life of both endogenous and ectopically added GLP-1 in thetreatment of disorders associated with obesity.

DPP IV inhibitors have hypoglycemic and antidiabetic activities, and canbe used in the treatment of disorders marked by aberrant glucosemetabolism (including storage). In particular embodiments, theinhibitors of the invention are useful as insulinotropic agents, or topotentiate the insulinotropic effects of such molecules as GLP-1. Inthis regard, the invention also provides methods for the treatmentand/or prophylaxis of a variety of disorders, including one or more of:hyperlipidemia, hyperglycemia, glucose tolerance insufficiency, insulinresistance and diabetic complications.

Another aspect of the invention features methods and pro-soft inhibitorcompounds for altering the pharmokinetics of a variety of differentpolypeptide hormones by inhibiting the proteolysis of one or morepeptide hormones by DPP IV or some other proteolytic activity. Forinstance, post-secretory metabolism is an important element in theoverall homeostasis of regulatory peptides, and the other enzymesinvolved in these processes may be suitable targets for pharmacologicalintervention by the subject method. In certain instances, the subjectmethod can be used to increase the half-life of otherproglucagon-derived peptides, such as glicentin (corresponding to PG1-69), oxyntomodulin (PG 33-69), glicentin-related pancreaticpolypeptide (GRPP, PG 1-30), intervening peptide-2 (IP-2, PG 111-122amide), and glucagon-like peptide-2 (GLP-2, PG 126-158). GLP-2, forexample, has been identified as a factor responsible for inducingproliferation of intestinal epithelium. See, e.g., Drucker et al. Proc.Natl. Acad. Sci. USA 1996, 93, 7911. The DPP IV inhibitors can also beused as part of a regimen for treating injury, inflammation or resectionof intestinal tissue, e.g., where enhanced growth and repair of theintestinal mucosal epithelial is desired, such as in the treatment ofCrohn's disease or Inflammatory Bowel Disease (IBD).

Another aspect of the invention relates to a method of treating growthhormone deficient children or improving nutrition or altering bodycomposition (muscle vs. fat) in adults. DPP IV has been implicated inthe metabolism and inactivation of growth hormone-releasing factor(GHRF). GHRF is a member of the family of homologous peptides thatincludes glucagon, secretin, vasoactive intestinal peptide (VIP),peptide histidine isoleucine (PHI), pituitary adenylate cyclaseactivating peptide (PACAP), gastric inhibitory peptide (GIP) andhelodermin. Kubiak et al. Peptide Res. 1994, 7, 153. GHRF is secreted bythe hypothalamus, and stimulates the release of growth hormone (GH) fromthe anterior pituitary. The subject method can also be used inveterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

The DPP IV inhibitors of the invention can be used to alter the plasmahalf-life of secretin, VIP, PHI, PACAP, GIP and/or helodermin. Incertain instances, the inhibitors can also be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, because DPP IV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity. In other embodiments, the DPP IV inhibitors can be used tostimulate hematopoiesis. In still other embodiments, the DPP IVinhibitors can be used to inhibit growth or vascularization oftransformed cells/tissues, e.g., to inhibit cell proliferation such asthat associated with tumor growth and metastasis, and for inhibitingangiogenesis in an abnormal proliferative cell mass. In yet otherembodiments, the subject DPP IV inhibitors can be used to reduceimmunological responses, e.g., as an immunosuppressant.

In yet other examples, the DPP IV inhibitors according to the presentinvention can be used to treat CNS maladies such as strokes, tumors,ischemia, Parkinson's disease, memory loss, hearing loss, vision loss,migraines, brain injury, spinal cord injury, Alzheimer's disease andamyotrophic lateral sclerosis (which has a CNS component). Additionally,the DPP IV inhibitors can be used to treat disorders having a moreperipheral nature, including multiple sclerosis and diabetic neuropathy.

Another aspect of the present invention provides a method forstimulating hematopoietic cells in culture or in vivo. In certainembodiments, the subject DPP IV pro-inhibitors include an address moietythat is a substrate for a protease that is expressed in bone marrow. TheDPP IV inhibitors of the invention can be used to restore or prevent adeficiency in hematopoietic cell number in a subject. Such deficienciescan arise, for example, from genetic abnormalities, disease, stress,chemotherapy, and radiation treatment.

Another aspect of the present invention features compounds which inhibitproteasome function. In certain embodiments, the pro-soft inhibitorsproduce inhibitor moieties that are potent and highly selectiveproteasome inhibitors and can be employed to inhibit proteasome functionInhibition of proteasome function has a number of practical therapeuticand prophylactic applications. For instance, the proteasomepro-inhibitors embodiments can include address moieties that aresubstrates for proteases that are expressed in tumors or other cellswhich are undergoing unwanted proliferation, or expressed in the tissuesurrounding the tumor or other target proliferating cells.

In certain embodiments, the proteasome pro-inhibitors of the presentinvention provide a method of reducing the rate of degradation of tumorsuppressors. In other embodiments, compounds of the present inventioninhibit the growth of cancer cells. In yet other embodiments, thecompounds of the present invention can be formulated in topical form fortreatment of skin disorders. Such pro-inhibitors are contemplated aspossessing important practical application in treating cellproliferative diseases, such as cancer, restenosis, and psoriasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts Ala-boroPro Thioxamide.

FIG. 2 depicts the pH-dependent behavior of Ala-boroPro Thioxamide andAla-boroPro.

FIG. 3 depicts a comparison of the IC₅₀ value of Ala-boroPro Thioxamideagainst DPP IV, DP8, and DP9. IC₅₀ values for Ala-boroPro Thioxamideinhibition of DPP IV, DP8 and DP9 were measured in 50 mM sodiumphosphate at pH 7.8 Inhibitor and enzyme were incubated at variousinhibitor concentrations prior to addition of the substrateAla-Pro-paranitroanalide at a concentration equal to the K_(m) for eachenzyme (20 μM for DPP IV and 100 μM for DP8 and DP9.) Reaction mixtureswere incubated at 37° C. for 30 min and then the absorbance at 410 nmwas read. Data were normalized to the uninhibited reaction rate for eachenzyme.

FIG. 4 depicts the K₁ of Ala-boroPro Thioxamide in an assay measuringinhibition of DPP IV. Purified DPP IV from human placenta was incubatedwith inhibitor in 50 mM HEPES, pH 8.0, 0.14 M NaCl at 23° C. to allowcomplete binding. The chromogenic substrate Ala-Pro-paranitroanalide wasadded to the enzyme inhibitor complex at 5 times the K_(m) value and thereaction was monitored by measuring the absorbance at 410 nm for 2 min.Enzyme concentration was determined independently. The Rate vs Inhibitorconcentration was fit to a simple equilibrium model to obtain the valueof K₁ (Gutheil and Bachovchin, 1993 Biochemistry 32(34) 8723-8731).

FIG. 5 depicts the results from an oral glucose challenge usingAla-boroPro Thioxamide. Following an overnight fast, seven C57BL/6 micewere given the drug or vehicle (0.25% methylcellulose), by oral gavage,two hours before an oral dose of 5 g/kg glucose. Blood was taken fromthe tail vein and glucose measured with a freestyle blood glucose meterjust before the drug, before glucose, and at 20, 40, 60 and 120 minafter the glucose. AUC was calculated for the data from zero to 120 min.

FIG. 6 depicts the results of rat plasma DPP IV inhibition usingAla-boroPro Thioxamide. Groups of four rats were given each dose ofAla-Pro thioxamide. Blood samples were taken from the tail vein andplasma DPP IV was measured by addition of 10 of plasma to 150 μL of 30μM Ala-Pro paranitroanalide in 50 mM HEPES, pH 8, 0.14 M NaCl. Thechange in absorbance at 410 nm was recorded after 1 hour. The data wasnormalized to the average pre-dose value for each group.

FIG. 7 depicts inactivation of Ala-boroPro Thioxamide at pH 7.8.Ala-boroPro thioxamide was pre-equilibrated at pH 2 and then the pHjumped to pH 7.8 by the addition of 0.6 M sodium phosphate buffer. ¹HNMR spectra were recorded as a function of time at pH 7.8. Twowell-resolved resonances representing the active (pH 2) and inactive (pH7.8) forms of the drug were integrated and the integral as a function oftime was fit to a single exponential. The same procedure was followedfor inactivation of Ala-boroPro. The half-life for the reaction was 60min for Ala-boroPro thioxamide and 30 min for Ala-boroPro.

FIG. 8 depicts inactivation of Ala-boroPro Thioxamide at pH 7.8.Ala-boroPro and Ala-boroPro thioxamide were pre-equilibrated at pH 2 andthen the pH jumped to pH 7.8 by the addition of 0.5 M sodium phosphatebuffer. DPP IV inhibition was measured at various times after the pHjump and the IC₅₀ values were plotted as a function of time. From theIC₅₀ value, the mole fraction of active inhibitor was calculatedassuming the pH 2 sample was 100% active. This value was fit to a singleexponential. Half-lives obtained from these fits were 47 min forAla-boroPro thioxamide and 24 min for Ala-boroPro.

FIG. 9 depicts results of ¹H NMR analysis of Ala-boroPro Thioxamide.

FIG. 10 depicts K_(i) measurements of Ala-boroPro Thioxamide.

FIG. 11 depicts a comparison of deactivation rates for Ala-boroPro andAla-boroPro Thioxamide.

FIG. 12 depicts OGTT with Ala-boroPro Thioxamide.

FIG. 13 depicts a comparison of Ala-boroPro and Ala-boroPro Thioxamideagainst DPPIV.

FIG. 14 depicts the activity of rat plasma DPP IV in the presence ofAla-boroPro Thioxamide.

FIG. 15 depicts Ala-boroPro Thioxamide, NVP LAF327, and MK0431.

FIG. 16 depicts the results of Ala-boroPro Thioxamide, NVP LAF327, andMK0431 in an assay measuring inhibition of rat plasma DPP IV.

FIG. 17 depicts the results of Ala-boroPro Thioxamide, NVP LAF327, andMK0431 in an assay measuring inhibition of glucose excursion.

FIG. 18 depicts K_(i) and IC₅₀ values for Ala-boroPro Thioxamide, NVPLAF327, and MK0431.

FIG. 19 depicts the results of Ala-boroPro Thioxamide, NVP LAF327, andMK0431 in an assay measuring rat plasma DPP IV activity following asingle oral dose (1 mg/kg).

FIG. 20 depicts the results of Ala-boroPro Thioxamide, NVP LAF327, andMK0431 in an assay measuring inhibition of rat plasma DPP IV.

FIG. 21 depicts OGTT with Ala-boroPro Thioxamide and LAF327.

FIG. 22 depicts the results of Ala-boroPro Thioxamide in an oral glucosechallenge assay in normal mice.

FIG. 23 depicts the results of NVP LAF327 in an oral glucose challengeassay in normal mice.

FIG. 24 depicts the results of MK0431 in an oral glucose challenge assayin normal mice. See Kim et al. J. Med. Chem. 2005, 48, 141-51.

FIG. 25 depicts the K₁ of AbP thioxamide and NVP LAF237.

FIG. 26 depicts the K₁ for MK0431.

FIG. 27 depicts binding/release of MK0431 with DPP IV. On-rate:Simultaneously mix enzyme, substrate and inhibitor. Curve responserepresents slow binding. Off-rate: Pre-incubate enzyme with inhibitor athigh concentration. Then, dilute mixture and add substrate. Curvereflects slow release of inhibitor following dilution.

FIG. 28 depicts certain compounds of the invention.

FIG. 29 depicts certain compounds of the invention.

FIG. 30 depicts certain compounds of the invention.

FIG. 31 depicts certain compounds of the invention.

FIG. 32 depicts certain compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION Overview

The present invention provides protease inhibitors and methods of usingprotease inhibitors. The invention features inhibitors for a wide arrayof proteases. For example, the protease may be a post-proline cleavingenzyme (PPCE), such as dipeptidyl peptidase IV. The invention alsoprovides compounds that inhibit proteasome activity. In certaininstances, the protease inhibitor is a pro-soft inhibitor. A pro-softinhibitor is an inactive agent that is activated, i.e., cleaved by an“activating protease,” to release an active inhibitor moiety inproximity to a “target protease.” The identity of the activatingprotease and target protease can be the same or different. Afteractivation of the pro-soft inhibitor, the active inhibitor moietyundergoes self-inactivation by proto-deboronation. In certain instances,the invention features dipeptide boronic acid inhibitors of the typeXaa-boroPro, where Xaa refers to any natural or non-naturally occurringamino acid comprising a thioxamide moiety, and boroPro refers to theanalog of proline in which the C-terminal carboxylate has been replacedby a boronyl group. Such compounds are potent inhibitors of dipeptidylamino peptidase type IV (DPP IV). The dipeptide boronic acid compoundsexist in open chain form under acidic conditions, but undergoproto-deboronation at neutral and basic conditions. The open chain formis active as an enzyme inhibitor; the compound formed from theproto-deboronation reaction is substantially inactive as an enzymeinhibitor. The pro-soft inhibitors of the present invention do notthemselves undergo proto-deboronation and can be constructed such thatthey do not inhibit the selected target enzyme, or other enzymes to anysignificant extent, before being cleaved by the activating protease.

One of the features that makes the pro-soft inhibitor molecules of thecurrent invention different from typical prodrugs is that the inhibitormoiety, after being generated in the active form near the target,undergoes inactivation over time, e.g., as it diffuses away from thetarget enzyme, thereby reducing the possibility of deleterious sideeffects that may result from inhibition of enzymes occurring in otherparts of the patient. This combination of being released in an activeform in the vicinity of the target enzyme together with this“programmed” deactivation mechanism makes the molecules of the inventionmore specific, effective, and safer (i.e., having fewer side effects)than the inhibitor moiety used on its own.

Advantageous features for compounds of the present invention include:better therapeutic indices, owing in part to reduced toxicity and/orimproved specificity for the targeted protease; better oralavailability; increased shelf-life; and/or increased duration of action(such as single oral dosage formulations which are effective for morethan 4 hours, and even more preferably for more than 8, 12, or 16hours). In certain instances, a compound of the invention has a K₁ forDPIV inhibition of about 50.0 nm or less, more preferably of about 10.0nm or less, and even more preferably of about 1.0, 0.1, or even 0.01 nMor less. Indeed, inhibitors with K₁ values in the picomolar and evenfemtomolar range are contemplated.

Another advantageous feature for compounds of the present invention isthat proto-deboronation irreversibly releases innocuous boric acid. TheLD₅₀ of boric acid is approximately equal to that of common table salt.Accordingly, long-term chronic therapy with the compounds of the presentinvention is expected to yield an improved safety profile (fewer sideeffects).

The compounds of the present invention can be used as part of treatmentsfor a variety of disorders/conditions, such as those which are mediatedby DPIV. For instance, the compounds can be used to up-regulate GIP andGLP-1 activities, e.g., by increasing the half-life of those hormones,as part of a treatment for regulating glucose levels and/or metabolism,e.g., to reduce insulin resistance, treat hyperglycemia,hyperinsulinemia, obesity, hyperlipidemia, hyperlipoproteinemia (such aschylomicrons, VLDL and LDL), and to regulate body fat and more generallylipid stores, and, more generally, for the improvement of metabolismdisorders, especially those associated with diabetes, obesity and/oratherosclerosis.

Certain of the subject compounds have extended duration. Accordingly, incertain certain embodiments, the compound is selected, and the amount ofcompound formulated, to provide a dosage which inhibits serum PPCE(e.g., DPIV) levels by at least 50% for at least 4 hours after a singledose, and even more preferably for at least 8 hours or even 12 or 16hours after a single dose.

For instance, in certain embodiments the method involves administrationof a DPIV inhibitor, preferably at a predetermined time(s) during a24-hour period, in an amount effective to improve one or more aberrantindices associated with glucose metabolism disorders (e.g., glucoseintolerance, insulin resistance, hyperglycemia, hyperinsulinemia, andType I and II diabetes).

In other embodiments, the method involves administration of a DPIVinhibitor in an amount effective to improve aberrant indices associatedwith obesity. Fat cells release the hormone leptin, which travels in thebloodstream to the brain and, through leptin receptors there, stimulatesproduction of GLP-1. GLP-1, in turn, produces the sensation of beingfull. The leading theory is that the fat cells of most obese peopleprobably produce enough leptin, but leptin may not be able to properlyengage the leptin receptors in the brain, and so does not stimulateproduction of GLP-1. There is accordingly a great deal of researchtowards utilizing preparations of GLP-1 as an appetite suppressant. Thesubject method provides a means for increasing the half-life of bothendogenous and ectopically added GLP-1 in the treatment of disordersassociated with obesity.

In a more general sense, the present invention provides methods andcompositions for altering the pharmacokinetics of a variety of differentpolypeptide hormones by inhibiting the proteolysis of one or morepeptide hormones by DPIV or some other proteolytic activity.Post-secretory metabolism is an important element in the overallhomeostasis of regulatory peptides, and the other enzymes involved inthese processes may be suitable targets for pharmacological interventionby the subject method. For example, the subject method can be used toincrease the half-life of other proglucagon-derived peptides, such asglicentin (corresponding to PG 1-69), oxyntomodulin (PG 33-69),glicentin-related pancreatic polypeptide (GRPP, PG 1-30), interveningpeptide-2 (IP-2, PG 111-122amide), and glucagon-like peptide-2 (GLP-2,PG 126-158).

GLP-2, for example, has been identified as a factor responsible forinducing proliferation of intestinal epithelium. See, for example,Drucker et al. Proc. Natl. Acad. Sci. USA 1996, 93, 7911. The subjectmethod can be used as part of a regimen for treating injury,inflammation or resection of intestinal tissue, e.g., where enhancedgrowth and repair of the intestinal mucosal epithelial is desired, suchas in the treatment of Crohn's disease or Inflammatory Bowel Disease(IBD).

DPIV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP), and helodermin (Kubiak et al. Peptide Res. 1994, 7, 153). GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

Likewise, the DPIV inhibitors of the subject invention can be used toalter the plasma half-life of secretin, VIP, PHI, PACAP, GIP, and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

In other embodiments, the compounds can be used to stimulatehematopoiesis. In still other embodiments, the compounds can be used toinhibit growth or vascularization of transformed cells/tissues, e.g., toinhibit cell proliferation such as that associated with tumor growth andmetastasis, and for inhibiting angiogenesis in an abnormal proliferativecell mass. In yet other embodiments, the compounds can be used to reduceimmunological responses, e.g., as an immunosuppressant.

In yet other examples, the DPIV inhibitors according to the presentinvention can be used to treat CNS maladies such as strokes, tumors,ischemia, Parkinson's disease, memory loss, hearing loss, vision loss,migraines, brain injury, spinal cord injury, Alzheimer's disease, andamyotrophic lateral sclerosis (which has a CNS component). Additionally,the DPIV inhibitors can be used to treat disorders having a moreperipheral nature, including multiple sclerosis and diabetic neuropathy.

Another aspect of the present invention relates to pharmaceuticalcompositions of the subject post-proline cleaving enzyme inhibitors,particularly DPIV inhibitors, and their uses in treating and/orpreventing disorders which can be improved by altering the homeostasisof peptide hormones. In a certain embodiment, the compounds havehypoglycemic and antidiabetic activities, and can be used in thetreatment of disorders marked by aberrant glucose metabolism (includingstorage). In particular embodiments, the compositions of the subjectmethods are useful as insulinotropic agents, or to potentiate theinsulinotropic effects of such molecules as GLP-1. In this regard,certain embodiments of the present compositions can be useful for thetreatment and/or prophylaxis of a variety of disorders, including one ormore of: hyperlipidemia, hyperglycemia, obesity, glucose toleranceinsufficiency, insulin resistance, and diabetic complications.

In certain instances, the compounds of the subject method are smallmolecules, e.g., with molecular weights less than 7500 amu, preferablyless than 5000 amu, and even more preferably less than 2000 or even lessthan 1000 amu. In certain embodiments, the compounds are orally active.

In certain instances, the compounds of the invention are used oncombination with one or more pharmaceutical agents. A large number ofpharmaceutical agents are known in the art and are amenable for use inthe pharmaceutical compositions of the invention. The term“pharmaceutical agent” includes without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of disease or illness; orsubstances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

Non-limiting examples of broad categories of useful pharmaceuticalagents include the following therapeutic categories: anabolic agents,antacids, anti-asthmatic agents, anti-cholesterolemic and anti-lipidagents, anti-coagulants, anti-convulsants, anti-diarrheals,anti-emetics, anti-infective agents, anti-inflammatory agents,anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesityagents, anti-pyretic and analgesic agents, anti-spasmodic agents,anti-thrombotic agents, anti-uricemic agents, anti-anginal agents,antihistamines, anti-tussives, appetite suppressants, biologicals,cerebral dilators, coronary dilators, decongestants, diuretics,diagnostic agents, erythropoietic agents, expectorants, gastrointestinalsedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ionexchange resins, laxatives, mineral supplements, mucolytic agents,neuromuscular drugs, peripheral vasodilators, psychotropics, sedatives,stimulants, thyroid and anti-thyroid agents, uterine relaxants,vitamins, and prodrugs.

More specifically, non-limiting examples of useful pharmaceutical agentsinclude the following therapeutic categories: analgesics, such asnonsteroidal anti-inflammatory drugs, opiate agonists and salicylates;antihistamines, such as H₁-blockers and H₂-blockers; anti-infectiveagents, such as anthelmintics, antianaerobics, antibiotics,aminoglycoside antibiotics, antifungal antibiotics, cephalosporinantibiotics, macrolide antibiotics, miscellaneous beta-lactamantibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamideantibiotics, tetracycline antibiotics, antimycobacterials,antituberculosis antimycobacterials, antiprotozoals, antimalarialantiprotozoals, antiviral agents, anti-retroviral agents, scabicides,and urinary anti-infectives; antineoplastic agents, such as alkylatingagents, nitrogen mustard alkylating agents, nitrosourea alkylatingagents, antimetabolites, purine analog antimetabolites, pyrimidineanalog antimetabolites, hormonal antineoplastics, naturalantineoplastics, antibiotic natural antineoplastics, and vinca alkaloidnatural antineoplastics; autonomic agents, such as anticholinergics,antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics,cholinergic agonist parasympathomimetics, cholinesterase inhibitorpara-sympathomimetics, sympatholytics, alpha-blocker sympatholytics,beta-blocker sympatholytics, sympathomimetics, and adrenergic agonistsympathomimetics; cardiovascular agents, such as antianginals,beta-blocker antianginals, calcium-channel blocker antianginals, nitrateantianginals, antiarrhythmics, cardiac glycoside antiarrhythmics, classI antiarrhythmics, class II antiarrhythmics, class III antiarrhythmics,class IV antiarrhythmics, antihypertensive agents, alpha-blockerantihypertensives, angiotensin-converting enzyme inhibitor (ACEinhibitor) antihypertensives, beta-blocker antihypertensives,calcium-channel blocker antihypertensives, central-acting adrenergicantihypertensives, diuretic antihypertensive agents, peripheralvasodilator antihypertensives, antilipemics, bile acid sequestrantantilipemics, HMG-CoA reductase inhibitor antilipemics, inotropes,cardiac glycoside inotropes, and thrombolytic agents; dermatologicalagents, such as antihistamines, anti-inflammatory agents, corticosteroidanti-inflammatory agents, antipruritics/local anesthetics, topicalanti-infectives, antifungal topical anti-infectives, antiviral topicalanti-infectives, and topical antineoplastics; electrolytic and renalagents, such as acidifying agents, alkalinizing agents, diuretics,carbonic anhydrase inhibitor diuretics, loop diuretics, osmoticdiuretics, potassium-sparing diuretics, thiazide diuretics, electrolytereplacements, and uricosuric agents; enzymes, such as pancreatic enzymesand thrombolytic enzymes; gastrointestinal agents, such asantidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents,salicylate gastrointestinal anti-inflammatory agents, antacid anti-ulceragents, gastric acid-pump inhibitor anti-ulcer agents, gastric mucosalanti-ulcer agents, H₂-blocker anti-ulcer agents, cholelitholytic agents,digestants, emetics, laxatives and stool softeners, and prokineticagents; general anesthetics, such as inhalation anesthetics, halogenatedinhalation anesthetics, intravenous anesthetics, barbiturate intravenousanesthetics, benzodiazepine intravenous anesthetics, and opiate agonistintravenous anesthetics; hematological agents, such as antianemiaagents, hematopoietic antianemia agents, coagulation agents,anticoagulants, hemostatic coagulation agents, platelet inhibitorcoagulation agents, thrombolytic enzyme coagulation agents, and plasmavolume expanders; hormones and hormone modifiers, such asabortifacients, adrenal agents, corticosteroid adrenal agents,androgens, anti-androgens, antidiabetic agents, sulfonylureaantidiabetic agents, antihypoglycemic agents, oral contraceptives,progestin contraceptives, estrogens, fertility agents, oxytocics,parathyroid agents, pituitary hormones, progestins, antithyroid agents,thyroid hormones, and tocolytics; immunobiologic agents, such asimmunoglobulins, immunosuppressives, toxoids, and vaccines; localanesthetics, such as amide local anesthetics and ester localanesthetics; musculoskeletal agents, such as anti-gout anti-inflammatoryagents, corticosteroid anti-inflammatory agents, gold compoundanti-inflammatory agents, immuno-suppressive anti-inflammatory agents,nonsteroidal anti-inflammatory drugs (NSAIDs), salicylateanti-inflammatory agents, skeletal muscle relaxants, neuromuscularblocker skeletal muscle relaxants, and reverse neuromuscular blockerskeletal muscle relaxants; neurological agents, such as anticonvulsants,barbiturate anticonvulsants, benzodiazepine anticonvulsants,anti-migraine agents, anti-parkinsonian agents, anti-vertigo agents,opiate agonists, and opiate antagonists; ophthalmic agents, such asanti-glaucoma agents, beta-blocker anti-gluacoma agents, mioticanti-glaucoma agents, mydriatics, adrenergic agonist mydriatics,antimuscarinic mydriatics, ophthalmic anesthetics, ophthalmicanti-infectives, ophthalmic aminoglycoside anti-infectives, ophthalmicmacrolide anti-infectives, ophthalmic quinolone anti-infectives,ophthalmic sulfonamide anti-infectives, ophthalmic tetracyclineanti-infectives, ophthalmic anti-inflammatory agents, ophthalmiccorticosteroid anti-inflammatory agents, and ophthalmic nonsteroidalanti-inflammatory drugs (NSAIDs); psychotropic agents, such asantidepressants, heterocyclic antidepressants, monoamine oxidaseinhibitors (MAOIs), selective serotonin re-uptake inhibitors (SSRIs),tricyclic antidepressants, antimanics, antipsychotics, phenothiazineantipsychotics, anxiolytics, sedatives, and hypnotics, barbituratesedatives and hypnotics, benzodiazepine anxiolytics, sedatives, andhypnotics, and psychostimulants; respiratory agents, such asantitussives, bronchodilators, adrenergic agonist bronchodilators,antimuscarinic bronchodilators, expectorants, mucolytic agents,respiratory anti-inflammatory agents, and respiratory corticosteroidanti-inflammatory agents; toxicology agents, such as antidotes, heavymetal antagonists/chelating agents, substance abuse agents, deterrentsubstance abuse agents, and withdrawal substance abuse agents; minerals;and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D,vitamin E, and vitamin K.

Preferred classes of useful pharmaceutical agents from the abovecategories include: (1) nonsteroidal anti-inflammatory drugs (NSAIDs)analgesics, such as diclofenac, ibuprofen, ketoprofen, and naproxen; (2)opiate agonist analgesics, such as codeine, fentanyl, hydromorphone, andmorphine; (3) salicylate analgesics, such as aspirin (ASA) (entericcoated ASA); (4) H₁-blocker antihistamines, such as clemastine andterfenadine; (5) H₂-blocker antihistamines, such as cimetidine,famotidine, nizadine, and ranitidine; (6) anti-infective agents, such asmupirocin; (7) antianaerobic anti-infectives, such as chloramphenicoland clindamycin; (8) antifungal antibiotic anti-infectives, such asamphotericin b, clotrimazole, fluconazole, and ketoconazole; (9)macrolide antibiotic anti-infectives, such as azithromycin anderythromycin; (10) miscellaneous beta-lactam antibiotic anti-infectives,such as aztreonam and imipenem; (11) penicillin antibioticanti-infectives, such as nafcillin, oxacillin, penicillin G, andpenicillin V; (12) quinolone antibiotic anti-infectives, such asciprofloxacin and norfloxacin; (13) tetracycline antibioticanti-infectives, such as doxycycline, minocycline, and tetracycline;(14) antituberculosis antimycobacterial anti-infectives such asisoniazid (INH), and rifampin; (15) antiprotozoal anti-infectives, suchas atovaquone and dapsone; (16) antimalarial antiprotozoalanti-infectives, such as chloroquine and pyrimethamine; (17)anti-retroviral anti-infectives, such as ritonavir and zidovudine; (18)antiviral anti-infective agents, such as acyclovir, ganciclovir,interferon alfa, and rimantadine; (19) alkylating antineoplastic agents,such as carboplatin and cisplatin; (20) nitrosourea alkylatingantineoplastic agents, such as carmustine (BCNU); (21) antimetaboliteantineoplastic agents, such as methotrexate; (22) pyrimidine analogantimetabolite antineoplastic agents, such as fluorouracil (5-FU) andgemcitabine; (23) hormonal antineoplastics, such as goserelin,leuprolide, and tamoxifen; (24) natural antineoplastics, such asaldesleukin, interleukin-2, docetaxel, etoposide (VP-16), interferonalfa, paclitaxel, and tretinoin (ATRA); (25) antibiotic naturalantineoplastics, such as bleomycin, dactinomycin, daunorubicin,doxorubicin, and mitomycin; (26) vinca alkaloid natural antineoplastics,such as vinblastine and vincristine; (27) autonomic agents, such asnicotine; (28) anticholinergic autonomic agents, such as benztropine andtrihexyphenidyl; (29) antimuscarinic anticholinergic autonomic agents,such as atropine and oxybutynin; (30) ergot alkaloid autonomic agents,such as bromocriptine; (31) cholinergic agonist parasympathomimetics,such as pilocarpine; (32) cholinesterase inhibitor parasympathomimetics,such as pyridostigmine; (33) alpha-blocker sympatholytics, such asprazosin; (34) beta-blocker sympatholytics, such as atenolol; (35)adrenergic agonist sympathomimetics, such as albuterol and dobutamine;(36) cardiovascular agents, such as aspirin (ASA) (enteric coated ASA);(37) beta-blocker antianginals, such as atenolol and propranolol; (38)calcium-channel blocker antianginals, such as nifedipine and verapamil;(39) nitrate antianginals, such as isosorbide dinitrate (ISDN); (40)cardiac glycoside antiarrhythmics, such as digoxin; (41) class Ianti-arrhythmics, such as lidocaine, mexiletine, phenyloin,procainamide, and quinidine; (42) class II antiarrhythmics, such asatenolol, metoprolol, propranolol, and timolol; (43) class IIIantiarrhythmics, such as amiodarone; (44) class IV antiarrhythmics, suchas diltiazem and verapamil; (45) alpha-blocker antihypertensives, suchas prazosin; (46) angiotensin-converting enzyme inhibitor (ACEinhibitor) antihypertensives, such as captopril and enalapril;(47).beta.-blocker antihypertensives, such as atenolol, metoprolol,nadolol, and propanolol; (48) calcium-channel blocker antihypertensiveagents, such as diltiazem and nifedipine; (49) central-acting adrenergicantihypertensives, such as clonidine and methyldopa; (50) diurecticantihypertensive agents, such as amiloride, furosemide,hydrochlorothiazide (HCTZ), and spironolactone; (51) peripheralvasodilator antihypertensives, such as hydralazine and minoxidil; (52)antilipemics, such as gemfibrozil and probucol; (53) bile acidsequestrant antilipemics, such as cholestyramine; (54) HMG-CoA reductaseinhibitor antilipemics, such as lovastatin and pravastatin; (55)inotropes, such as aminone, dobutamine, and dopamine; (56) cardiacglycoside inotropes, such as digoxin; (57) thrombolytic agents, such asalteplase (TPA), anistreplase, streptokinase, and urokinase; (58)dermatological agents, such as colchicine, isotretinoin, methotrexate,minoxidil, tretinoin (ATRA); (59) dermatological corticosteroidanti-inflammatory agents, such as betamethasone and dexamethasone; (60)antifungal topical anti-infectives, such as amphotericin B,clotrimazole, miconazole, and nystatin; (61) antiviral topicalanti-infectives, such as acyclovir; (62) topical antineoplastics, suchas fluorouracil (5-FU); (63) electrolytic and renal agents, such aslactulose; (64) loop diuretics, such as furosemide; (65)potassium-sparing diuretics, such as triamterene; (66) thiazidediuretics, such as hydro-chlorothiazide (HCTZ); (67) uricosuric agents,such as probenecid; (68) enzymes such as RNase and DNase; (69)thrombolytic enzymes, such as alteplase, anistreplase, streptokinase andurokinase; (70) antiemetics, such as prochlorperazine; (71) salicylategastrointestinal anti-inflammatory agents, such as sulfasalazine; (72)gastric acid-pump inhibitor anti-ulcer agents, such as omeprazole; (73)H₂-blocker anti-ulcer agents, such as cimetidine, famotidine,nizatidine, and ranitidine; (74) digestants, such as pancrelipase; (75)prokinetic agents, such as erythromycin; (76) opiate agonist intravenousanesthetics such as fentanyl; (77) hematopoietic antianemia agents, suchas erythropoietin, filgrastim (G-CSF), and sargramostim (GM-CSF); (78)coagulation agents, such as antihemophilic factors 1-10 (AHF 1-10); (79)anticoagulants, such as warfarin; (80) thrombolytic enzyme coagulationagents, such as alteplase, anistreplase, streptokinase and urokinase;(81) hormones and hormone modifiers, such as bromocriptine; (82)abortifacients, such as methotrexate; (83) antidiabetic agents, such asinsulin; (84) oral contraceptives, such as estrogen and progestin; (85)progestin contraceptives, such as levonorgestrel and norgestrel; (86)estrogens such as conjugated estrogens, diethylstilbestrol (DES),estrogen (estradiol, estrone, and estropipate); (87) fertility agents,such as clomiphene, human chorionic gonadatropin (HCG), and menotropins;(88) parathyroid agents such as calcitonin; (89) pituitary hormones,such as desmopressin, goserelin, oxytocin, and vasopressin (ADH); (90)progestins, such as medroxyprogesterone, norethindrone, andprogesterone; (91) thyroid hormones, such as levothyroxine; (92)immunobiologic agents, such as interferon beta-1b and interferongamma-1b; (93) immunoglobulins, such as immune globulin 1M, IMIG, IGIMand immune globulin IV, IVIG, IGIV; (94) amide local anesthetics, suchas lidocaine; (95) ester local anesthetics, such as benzocaine andprocaine; (96) musculoskeletal corticosteroid anti-inflammatory agents,such as beclomethasone, betamethasone, cortisone, dexamethasone,hydrocortisone, and prednisone; (97) musculoskeletal anti-inflammatoryimmunosuppressives, such as azathioprine, cyclophosphamide, andmethotrexate; (98) musculoskeletal nonsteroidal anti-inflammatory drugs(NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketorlac, andnaproxen; (99) skeletal muscle relaxants, such as baclofen,cyclobenzaprine, and diazepam; (100) reverse neuromuscular blockerskeletal muscle relaxants, such as pyridostigmine; (101) neurologicalagents, such as nimodipine, riluzole, tacrine and ticlopidine; (102)anticonvulsants, such as carbamazepine, gabapentin, lamotrigine,phenyloin, and valproic acid; (103) barbiturate anticonvulsants, such asphenobarbital and primidone; (104) benzodiazepine anticonvulsants, suchas clonazepam, diazepam, and lorazepam; (105) anti-parkisonian agents,such as bromocriptine, levodopa, carbidopa, and pergolide; (106)anti-vertigo agents, such as meclizine; (107) opiate agonists, such ascodeine, fentanyl, hydromorphone, methadone, and morphine; (108) opiateantagonists, such as naloxone; (109).beta.-blocker anti-glaucoma agents,such as timolol; (110) miotic anti-glaucoma agents, such as pilocarpine;(111) ophthalmic aminoglycoside antiinfectives, such as gentamicin,neomycin, and tobramycin; (112) ophthalmic quinolone anti-infectives,such as ciprofloxacin, norfloxacin, and ofloxacin; (113) ophthalmiccorticosteroid anti-inflammatory agents, such as dexamethasone andprednisolone; (114) ophthalmic nonsteroidal anti-inflammatory drugs(NSAIDs), such as diclofenac; (115) antipsychotics, such as clozapine,haloperidol, and risperidone; (116) benzodiazepine anxiolytics,sedatives and hypnotics, such as clonazepam, diazepam, lorazepam,oxazepam, and prazepam; (117) psychostimulants, such as methylphenidateand pemoline; (118) antitussives, such as codeine; (119)bronchodilators, such as theophylline; (120) adrenergic agonistbronchodilators, such as albuterol; (121) respiratory corticosteroidanti-inflammatory agents, such as dexamethasone; (122) antidotes, suchas flumazenil and naloxone; (123) heavy metal antagonists/chelatingagents, such as penicillamine; (124) deterrent substance abuse agents,such as disulfuram, naltrexone, and nicotine; (125) withdrawal substanceabuse agents, such as bromocriptine; (126) minerals, such as iron,calcium, and magnesium; (127) vitamin B compounds, such ascyanocobalamin (vitamin B12) and niacin (vitamin B3); (128) vitamin Ccompounds, such as ascorbic acid; and (129) vitamin D compounds, such ascalcitriol.

In addition to the foregoing, the following less common drugs may alsobe used: chlorhexidine; estradiol cypionate in oil; estradiol valeratein oil; flurbiprofen; flurbiprofen sodium; ivermectin; levodopa;nafarelin; and somatropin. Further, the following drugs may also beused: recombinant beta-glucan; bovine immunoglobulin concentrate; bovinesuperoxide dismutase; the formulation comprising fluorouracil,epinephrine, and bovine collagen; recombinant hirudin (r-Hir), HIV-1immunogen; human anti-TAC antibody; recombinant human growth hormone(r-hGH); recombinant human hemoglobin (r-Hb); recombinant humanmecasermin (r-IGF-1); recombinant interferon beta-1a; lenograstim(G-CSF); olanzapine; recombinant thyroid stimulating hormone (r-TSH);and topotecan.

Further still, the following intravenous products may be used: acyclovirsodium; aldesleukin; atenolol; bleomycin sulfate, human calcitonin;salmon calcitonin; carboplatin; carmustine; dactinomycin, daunorubicinHCl; docetaxel; doxorubicin HCl; epoetin alfa; etoposide (VP-16);fluorouracil (5-FU); ganciclovir sodium; gentamicin sulfate; interferonalfa; leuprolide acetate; meperidine HCl; methadone HCl; methotrexatesodium; paclitaxel; ranitidine HCl; vinblastin sulfate; and zidovudine(AZT).

Further specific examples of useful pharmaceutical agents from the abovecategories include: (a) anti-neoplastics such as androgen inhibitors,antimetabolites, cytotoxic agents, and immunomodulators; (b)anti-tussives such as dextromethorphan, dextromethorphan hydrobromide,noscapine, carbetapentane citrate, and chlorphedianol hydrochloride; (c)antihistamines such as chlorpheniramine maleate, phenindamine tartrate,pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate;(d) decongestants such as phenylephrine hydrochloride,phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, andephedrine; (e) various alkaloids such as codeine phosphate, codeinesulfate and morphine; (f) mineral supplements such as potassiumchloride, zinc chloride, calcium carbonates, magnesium oxide, and otheralkali metal and alkaline earth metal salts; (g) ion exchange resinssuch as cholestryramine; (h) anti-arrhythmics such asN-acetylprocainamide; (i) antipyretics and analgesics such asacetaminophen, aspirin and ibuprofen; (j) appetite suppressants such asphenyl-propanolamine hydrochloride or caffeine; (k) expectorants such asguaifenesin; (l) antacids such as aluminum hydroxide and magnesiumhydroxide; (m) biologicals such as peptides, polypeptides, proteins andamino acids, hormones, interferons or cytokines, and other bioactivepeptidic compounds, such as interleukins 1-18 including mutants andanalogues, RNase, DNase, luteinizing hormone releasing hormone (LHRH)and analogues, gonadotropin releasing hormone (GnRH), transforminggrowth factor-beta (TGF-beta), fibroblast growth factor (FGF), tumornecrosis factor-alpha & beta (TNF-alpha & beta), nerve growth factor(NGF), growth hormone releasing factor (GHRF), epidermal growth factor(EGF), fibroblast growth factor homologous factor (FGFHF), hepatocytegrowth factor (HGF), insulin growth factor (IGF), invasion inhibitingfactor-2 (IIF-2), bone morphogenetic proteins 1-7 (BMP 1-7),somatostatin, thymosin-alpha-1, gamma-globulin, superoxide dismutase(SOD), complement factors, hGH, tPA, calcitonin, ANF, EPO and insulin;and (n) anti-infective agents such as antifungals, anti-virals,antiseptics and antibiotics.

Alternatively, the pharmaceutical agent may be a radiosensitizer, suchas metoclopramide, sensamide or neusensamide (manufactured by Oxigene);profiromycin (made by Vion); RSR13 (made by Allos); Thymitaq (made byAgouron), etanidazole or lobenguane (manufactured by Nycomed);gadolinium texaphrin (made by Pharmacyclics); BuDR/Broxine (made byNeoPharm); IPdR (made by Sparta); CR2412 (made by Cell Therapeutic); L1X(made by Terrapin); or the like. Preferably, the biologically activesubstance is selected from the group consisting of the group consistingof peptides, poly-peptides, proteins, amino acids, polysaccharides,growth factors, hormones, anti-angiogenesis factors, interferons orcytokines, and pro-drugs. In a particularly certain embodiment, thebiologically active substance is a therapeutic drug or pro-drug, mostpreferably a drug selected from the group consisting of chemotherapeuticagents and other anti-neoplastics such as paclitaxel, antibiotics,anti-virals, antifungals, anti-inflammatories, and anticoagulants.

The biologically active substances are used in amounts that aretherapeutically effective. While the effective amount of a biologicallyactive substance will depend on the particular material being used,amounts of the biologically active substance from about 1% to about 65%may be desirable. Lesser amounts may be used to achieve efficaciouslevels of treatment for certain biologically active substances.

DEFINITIONS

The term “high affinity” as used herein means strong binding affinitybetween molecules with a dissociation constant K_(D) of no greater than1 μM. In a preferred case, the K_(D) is less than 100 nM, 10 nM, 1 nM,100 μM, or even 10 μM or less. In a most certain embodiment, the twomolecules can be covalently linked (K_(D) is essentially 0).

The term “boro-Ala” refers to the analog of alanine in which thecarboxylate group (COOH) is replaced with a boronyl group (B(OH)₂).Likewise, the term “boro-Pro” refers to the analog of proline in whichthe carboxylate group (COOH) is replaced with a boronyl group (B(OH)₂).More generally, the term “boro-Xaa”, where Xaa is an amino acid residue,refers to the analog of an amino acid in which the carboxylate group(COOH) is replaced with a boronyl group (B(OH)₂).

The term “Ala-boroPro” refers to

The term “Ala-boroPro thioxo amide” refers to

The term “Pro-boroPro” refers to

The term “thioxam” used in association with chemical nomenclature refersto a compound wherein at least one amide group has been replaced by atleast one thioxamide group. For example Pro(thioxam) refers to a prolineresidue wherein the amide group has been replaced by a thioxamide group.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human subject. Non-human subjects include farmanimals (e.g., cows, horses, pigs, sheep) and companion animals (e.g.,cats, dogs).

The term “ED₅₀” means the dose of a drug that, in 50% of patients, willprovide a clinically relevant improvement or change in a physiologicalmeasurement, such as glucose responsiveness, increase in hematocrit,decrease in tumor volume, etc.

The term “IC₅₀” means the dose of a drug that inhibits a biologicalactivity by 50%, e.g., the amount of compound required to inhibit atleast 50% of DPIV (or other PPCE) activity in vivo.

A compound is said to have an “insulinotropic activity” if it is able tostimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin.

The term “interact” as used herein is meant to include all interactions(e.g., biochemical, chemical, or biophysical interactions) betweenmolecules, such as protein-protein, protein-nucleic acid, nucleicacid-nucleic acid, protein-small molecule, nucleic acid-small molecule,or small molecule-small molecule interactions.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, delays the onset of, orotherwise inhibits symptoms of a medical condition in a subject relativeto a subject which does not receive the composition. Thus, prevention ofcancer includes, for example, reducing the number of detectablecancerous growths in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/or delayingthe appearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

A “therapeutically effective amount” of a compound, e.g., such as a DPIVinhibitor of the present invention, with respect to the subject methodof treatment, refers to an amount of the compound(s) in a preparationwhich, when administered as part of a desired dosage regimen (to amammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

A “single oral dosage formulation” is a dosage which provides an amountof drug to produce a serum concentration at least as great as the EC₅₀for that drug, but less than the LD₅₀. Another measure for a single oraldosage formulation is that it provides an amount of drug necessary toproduce a serum concentration at least as great as the IC₅₀ for thatdrug, but less than the LD₅₀. By either measure, a single oral dosageformulation is preferably an amount of drug which produces a serumconcentration at least 10% less than the LD₅₀, and even more preferablyat least 50%, 75%, or even 90% less than the drug's the LD₅₀.

An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyldefined below. A straight aliphatic chain is limited to unbranchedcarbon chain moieties. As used herein, the term “aliphatic group” refersto a straight chain, branched-chain, or cyclic aliphatic hydrocarbongroup and includes saturated and unsaturated aliphatic groups, such asan alkyl group, an alkenyl group, or an alkynyl group.

“Alkyl” refers to a fully saturated cyclic or acyclic, branched orunbranched carbon chain moiety having the number of carbon atomsspecified, or up to 30 carbon atoms if no specification is made. Forexample, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and thosemoieties which are positional isomers of these moieties. Alkyl of 10 to30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6, or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl,” asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths. Throughout the application,preferred alkyl groups are lower alkyls. In certain embodiments, asubstituent designated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —(S)-alkyl, —(S)-alkenyl,—(S)-alkynyl, and —(S)—(CH₂)_(m)—R¹, wherein m and R¹ are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

“Alkenyl” refers to any cyclic or acyclic, branched or unbranchedunsaturated carbon chain moiety having the number of carbon atomsspecified, or up to 26 carbon atoms if no limitation on the number ofcarbon atoms is specified; and having one or more double bonds in themoiety. Alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl,heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl,tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl,nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, andtetracosenyl, in their various isomeric forms, where the unsaturatedbond(s) can be located anywherein the moiety and can have either the (Z)or the (E) configuration about the double bond(s).

“Alkynyl” refers to hydrocarbyl moieties of the scope of alkenyl, buthaving one or more triple bonds in the moiety.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls, or alkynyls.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen moiety attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propoxy,tert-butoxy, and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R¹,where m and R₁ are described below.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the formulae:

wherein R³, R⁵ and R⁶ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R¹, or R³ and R⁵ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R¹ represents an alkenyl, aryl, cycloalkyl,a cycloalkenyl, a heterocyclyl, or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In certain embodiments, only one of R³or R⁵ can be a carbonyl, e.g., R³, R⁵, and the nitrogen together do notform an imide. In even more certain embodiments, R³ and R⁵ (andoptionally R⁶) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R¹. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₃ and R₅ isan alkyl group. In certain embodiments, an amino group or an alkylamineis basic, meaning it has a conjugate acid with a pK_(a)≧7.00, i.e., theprotonated forms of these functional groups have pK_(a)s relative towater above about 7.00.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon (i.e., carbocyclic aryl) or where one or moreatoms are heteroatoms (i.e., heteroaryl). The term “aryl” also includespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Carboycyclic aryl groups include benzene, naphthalene, phenanthrene,phenol, aniline, and the like. Heteroaryl groups include substituted orunsubstituted aromatic 5- to 7-membered ring structures, more preferably5- to 6-membered rings, whose ring structures include one to fourheteroatoms. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine and pyrimidine, and the like.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the formula:

wherein X is a bond or represents an oxygen or a sulfur, and R⁷represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R¹ or apharmaceutically acceptable salt, R⁸ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R¹, where m and R¹ are as defined above. Where Xis an oxygen and R⁷ or R⁸ is not hydrogen, the formula represents an“ester.” Where X is an oxygen, and R⁷ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R⁷ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R⁸ is a hydrogen, the formula represents a “formate.” Ingeneral, where the oxygen atom of the above formula is replaced by asulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R⁷ or R⁸ is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R⁷ is a hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R⁸ isa hydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R⁷ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R⁷ is a hydrogen,the above formula represents an “aldehyde” group.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN,and the like.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The term “hydrocarbyl” refers to a monovalent hydrocarbon moietycomprised of carbon chains or rings of up to 26 carbon atoms to whichhydrogen atoms are attached. The term includes alkyl, cycloalkyl,alkenyl, alkynyl, and aryl groups, groups which have a mixture ofsaturated and unsaturated bonds, carbocyclic rings, and includescombinations of such groups. It may refer to straight chain,branched-chain, cyclic structures, or combinations thereof.

The term “hydrocarbylene” refers to a divalent hydrocarbyl moiety.Representative examples include alkylene, phenylene, or cyclohexylene.Preferably, the hydrocarbylene chain is fully saturated and/or has achain of 1 to 10 carbon atoms.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” means —SH; theterm “hydroxyl” means —OH; the term “sulfonyl” means —SO₂—; the term“azido” means —N₃; the term “cyano” means —CN; the term “isocyanato”means —NCO; the term “thiocyanato” means —SCN; the term “isothiocyanato”means —NCS; and the term “cyanato” means —OCN.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the formula:

in which R³ and R⁵ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the formula:

in which R⁷ is as defined above.

The term “sulfonamide” is art recognized and includes a moiety that canbe represented by the formula:

in which R³ and R⁸ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the formula:

in which R⁷ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the formula:

in which R¹² is selected from the group consisting of the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aralkyl, or aryl.

The term “thioxamide,” as used herein, refers to a moiety that can berepresented by the formula:

in which R^(t) is selected from the group consisting of the groupconsisting of hydrogen, alkyl, cycloalkyl, aralkyl, or aryl, preferablyhydrogen or alkyl. Moreover, “thioxamide-derived” compounds or“thioxamide analogs” refer to compounds in which one or more amidegroups have been replaced by one or more corresponding thioxamidegroups. Thioxamides are also referred to in the art as “thioamides.”

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition else wherein the same structure.

“Biohydrolyzable amide” refers to an amide moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

“Biohydrolyzable ester” refers to an ester moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

“Biohydrolyzable imide” refers to an imide moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

The terms “amino acid residue” and “peptide residue” mean an amino acidor peptide molecule without the —OH of its carboxyl group. In generalthe abbreviations used herein for designating the amino acids and theprotective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature (see Biochemistry 1972, 11,1726-1732). For instance, Met, Ile, Leu, Ala, and Gly represent“residues” of methionine, isoleucine, leucine, alanine, and glycine,respectively. Residue means a moiety derived from the correspondingα-amino acid by eliminating the OH portion of the carboxyl group and theH portion of the α-amino group. The term “amino acid side chain” is thatpart of an amino acid exclusive of the —CH(NH₂)COOH portion, as definedby K. D. Kopple, Peptides and Amino Acids; Benjamin: N.Y., 1966; pp. 2and 33; examples of such side chains of the common amino acids are—CH₂CH₂SCH₃ (the side chain of methionine), —CH₂(CH₃)—CH₂CH₃ (the sidechain of isoleucine), —CH₂CH(CH₃)₂ (the side chain of leucine) or H-(theside chain of glycine).

For the most part, the amino acids used in the application of thisinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids which contain amino and carboxyl groups. Particularly suitableamino acid side chains include side chains selected from those of thefollowing amino acids: glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan, and those amino acids and aminoacid analogs which have been identified as constituents ofpeptidylglycan bacterial cell walls.

The term amino acid residue further includes analogs, derivatives andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g.,modified with an N-terminal or C-terminal protecting group). Forexample, the present invention contemplates the use of amino acidanalogs wherein a side chain is lengthened or shortened while stillproviding a carboxyl, amino or other reactive precursor functional groupfor cyclization, as well as amino acid analogs having variant sidechains with appropriate functional groups). For instance, the subjectcompound can include an amino acid analog such as, for example,cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine,homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan,1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, ordiaminobutyric acid. Other naturally occurring amino acid metabolites orprecursors having side chains which are suitable herein will berecognized by those skilled in the art and are included in the scope ofthe present invention.

Also included are the (D) and (L) stereoisomers of such amino acids whenthe structure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols (D), (L) or (DL), furthermore whenthe configuration is not designated, the amino acid or residue can havethe configuration (D), (L), or (DL). It will be noted that the structureof some of the compounds of this invention includes asymmetric carbonatoms. It is to be understood accordingly that the isomers arising fromsuch asymmetry are included within the scope of this invention. Suchisomers can be obtained in substantially pure form by classicalseparation techniques and by sterically controlled synthesis. For thepurposes of this application, unless expressly noted to the contrary, anamed amino acid shall be construed to include both the (D) and (L)stereoisomers.

The phrase “protecting group” as used herein means substituents whichprotect the reactive functional group from undesirable chemicalreactions. Examples of such protecting groups include esters ofcarboxylic acids and boronic acids, ethers of alcohols, and acetals andketals of aldehydes and ketones. For instance, the phrase “N-terminalprotecting group” or “amino-protecting group” as used herein refers tovarious amino-protecting groups which can be employed to protect theN-terminus of an amino acid or peptide against undesirable reactionsduring synthetic procedures. Examples of suitable groups include acylprotecting groups such as, to illustrate, formyl, dansyl, acetyl,benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromaticurethane protecting groups as, for example, benzyloxycarbonyl (Cbz); andaliphatic urethane protecting groups such as t-butoxycarbonyl (Boc) or9-Fluorenylmethoxycarbonyl (Fmoc).

As noted above, certain compounds of the present invention may exist inparticular geometric or stereoisomeric forms. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.In certain embodiments where a particular enantiomer is preferred, acompound of the present invention is enriched tohave >60%, >70%, >80%, >90%, >95%, or even greater than 98% or 99% ofthe preferred enantiomer, as opposed to a racemate where the twoenantiomers each are present to the extent of 50%.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomer. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomer.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th ed., 1986-87, inside cover.

A compound is said to have an “insulinotropic activity” if it is able tostimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin.

The term “amino-terminal protecting group” as used herein, refers toterminal amino protecting groups that are typically employed in organicsynthesis, especially peptide synthesis. Any of the known categories ofprotecting groups can be employed, including acyl protecting groups,such as acetyl, and benzoyl; aromatic urethane protecting groups, suchas benzyloxycarbonyl; and aliphatic urethane protecting groups, such astert-butoxycarbonyl. See, for example, Gross and Mienhoffer, Eds., ThePeptides, Academic Press: New York, 1981; Vol. 3, 3-88; and Green, T.W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed,Wiley: New York, 1991. Preferred protecting groups include aryl-,aralkyl-, heteroaryl- and heteroarylalkyl-carbonyl and sulfonylmoieties.

The term “amino acid analog” refers to a compound structurally similarto a naturally occurring amino acid wherein either the C-terminalcarboxy group, the N-terminal amino group or side-chain functional grouphas been chemically modified. For example, aspartic acid-(beta-methylester) is an amino acid analog of aspartic acid; N-ethylglycine is anamino acid analog of glycine; or alanine carboxamide is an amino acidanalog of alanine.

The terms “gastrointestinal inflammation,” “inflammatory bowel disease,”and “inflammation of the gastrointestinal tract” are usedinterchangeably herein to mean inflammation of any portion of thegastrointestinal tract, from the esophagus to the sigmoid flexure or thetermination of the colon in the rectum. The inflammation can be acute,but, generally, the composition of this invention is used to treatCrohnic conditions.

A “single oral dosage formulation” is a dosage which provides an amountof drug to produce a serum concentration at least as great as the EC₅₀for that drug, but less than the LD₅₀. Another measure for a single oraldosage formulation is that it provides an amount of drug necessary toproduce a serum concentration at least as great as the IC₅₀ for thatdrug, but less than the LD₅₀. By either measure, a single oral dosageformulation is preferably an amount of drug which produces a serumconcentration at least 10% less than the LD₅₀, and even more preferablyat least 50%, 75%, or even 90% less than the LD₅₀.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the inhibitors of thepresent invention from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) RingeRssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically acceptable salts” in these instances refersto the relatively non-toxic, inorganic and organic base addition saltsof compounds of the present invention.

The term “pharmaceutically functional derivative” refers to anypharmaceutically acceptable derivative of an inhibitor of the presentinvention, for example, an ester or an amide, which upon administrationto a mammal is capable of providing (directly or indirectly) theinhibitor. Such derivatives are recognizable to those skilled in theart, without undue experimentation. Nevertheless reference is made tothe teaching of Burger's Medicinal Chemistry and Drug Discovery, 5thed., Vol 1.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable mammalian cell

The term “prodrug” as used herein encompasses compounds that, underphysiological conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties that are hydrolyzed under physiological conditions to revealthe desired molecule. In other embodiments, the prodrug is converted byan enzymatic activity of the host animal.

The term “shelf-life” typically refers to the time period for which theperformance characteristics of an inhibitor remain at peak. As usedherein, the term “T₉₀” refers to the amount of time it takes for apreparation of the subject inhibitor to degrade to the point that it has90% of the activity of the starting sample, e.g., a diminishment of 10%.Likewise, the term “T₅₀” refers to the amount of time it takes for apreparation of the subject inhibitor to degrade to the point that it has50% of the activity of the starting sample, e.g., a diminishment of 50%.The shelf-life, whether reported as T₉₀ or T₅₀, for a givenpharmaceutical preparation of an inhibitor is the measured for thepreparation as it is packaged for use by a healthcare provider orpatient.

As used herein the term “substantially soluble” refers to inhibitorswhich can be dissolved in inhalant propeller mixture to form asubstantially clear to hazy solution which will not separate into layersor form a precipitate when left unagitated for a minimum of 24 hours atroom temperature.

By “transdermal patch” is meant a system capable of delivery of a drugto a patient via the skin, or any suitable external surface, includingmucosal membranes, such as those found inside the mouth. Such deliverysystems generally comprise a flexible backing, an adhesive and a drugretaining matrix, the backing protecting the adhesive and matrix and theadhesive holding the whole on the skin of the patient. On contact withthe skin, the drug-retaining matrix delivers inhibitor to the skin, thedrug then passing through the skin into the patient's system.

The term “quaternizing agent” refers to a chemical compound whichconverts a nitrogen atom with fewer than four substituents to apositively charged nitrogen atom with four substituents. Examples of“quaternizing agents” include lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides, and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethylbromides, and others.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

A “therapeutically effective amount” of a compound, e.g., such as adipeptidyl peptidase inhibitor of the present invention, with respect tothe subject method of treatment, refers to an amount of the compound(s)in a preparation which, when administered as part of a desired dosageregimen (to a mammal, preferably a human) brings alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated or the cosmetic purpose, e.g., at a reasonablebenefit/risk ratio applicable to any medical treatment.

A “therapeutically effective daily dosage” of a compound, e.g., such asan inhibitor of the present invention, with respect to the subjectmethod of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired daily dosageregimen (to a mammal, preferably a human) brings alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated or the cosmetic purpose, e.g., at a reasonablebenefit/risk ratio applicable to any medical treatment.

It will be understood that all generic structures recited herein, withrespect to appropriate combinations of substituents, are intended tocover those embodiments permitted by valency and stability.

EXEMPLARY EMBODIMENTS

(i). Compounds

Useful compounds will be described below using various formulae. In eachcase, the variables in the formula are defined specifically for eachindividual formulae. A definition of a variable for one formula shouldnot be used to vary a definition provided for another formula, althougha variable that has not been defined for one formula may be interpretedby analogy with a definition elsewhere for a similar formula.

Embodiment A

A representative class of compounds for use in the method of the presentinvention are represented by formula I:

wherein

A represents a 4-8 membered heterocycle including the N and the Cαcarbon;

Z represents C or N;

W represents a functional group which reacts with an active site residueof the targeted protease, as for example, —CN, —CH═NR₅,

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or

wherein the bond between R₁ and N is a thioxamide bond;

R₂ is absent or represents one or more substitutions to the ring A, eachof which is independently a halogen, lower alkyl, lower alkenyl, loweralkynyl, carbonyl (such as a carboxyl, ester, formate, or ketone),thiocarbonyl (such as a thioester, thioacetate, or thioformate), amino,acylamino, amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R₇,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R₇, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

when Z is N, R₃ is absent;

when Z is C, R₃ represents hydrogen or a halogen, lower alkyl, loweralkenyl, lower alkynyl, carbonyl, thiocarbonyl, amino, acylamino, amido,nitro, sulfate, sulfonate, a sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl, —(CH₂)₁,—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R₇, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

R₅ represents H, alkyl, alkenyl, alkynyl, —C(X₁)(X₂)X₃, —(CH₂)_(m)—R₇,—(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl, —(CH₂)_(n)—O-alkenyl,—(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(n)—SH,—(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇, —C(O)C(O)NH₂, or —C(O)C(O)OR′₇;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—-(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R′₇ represents, for each occurrence, hydrogen, or a substituted orunsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl,or heterocycle; and

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇;

R₅₂ represents hydrogen, a lower alkyl, amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain embodiments, the ring A is a 5-, 6-, or 7-membered ring,e.g., represented by

and more preferably a 5 or 6 membered ring. The ring may, optionally, befurther substituted.

In certain embodiments, W represents

In certain embodiments, R₁ is

wherein R₃₆ is a small hydrophobic group, e.g., a lower alkyl or ahalogen and R₃₈ is hydrogen, or, R₃₆ and R₃₈ together form a 4-7membered heterocycle including the N and the Cα carbon, as defined for Aabove; and R₄₀ represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group.

In certain embodiments, R₂ is absent, or represents a small hydrophobicgroup such as a lower alkyl or a halogen.

In certain embodiments, R₃ is a hydrogen, or a small hydrophobic groupsuch as a lower alkyl or a halogen.

In certain embodiments, R₅ is a hydrogen, or a halogenated lower alkyl.

In certain embodiments, X₁ is a fluorine, and X₂ and X₃, if halogens,are fluorine.

Also deemed as equivalents are any compounds which can be hydrolyticallyconverted into any of the aforementioned compounds including boronicacid esters and halides, and carbonyl equivalents including acetals,hemiacetals, ketals, and hemiketals, and cyclic dipeptide analogs.

Longer peptide sequences are needed for the inhibition of certainproteases and improve the specificity of the inhibition in some cases.

In certain embodiments, the subject method utilizes, as a DPIVinhibitor, a boronic acid analog of an amino acid or amino acidderivative, such as a thioxamide-modified amino acid. For example, thepresent invention contemplates the use of boro-prolyl derivatives in thesubject method. Exemplary boronic acid derived inhibitors of the presentinvention are represented by the formula II:

wherein

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or

wherein the bond between R₁ and N is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

or R₈ and R₉ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure;

R₁₁ and R₁₂ each independently represent hydrogen, a alkyl, or apharmaceutically acceptable salt, or R₁₁ and R₁₂ taken together with theO—B—O atoms to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In other embodiments, compounds include aldehyde analogs of proline orprolyl derivatives, such as thioxamide derivatives. Exemplaryaldehyde-derived compounds of the present invention are represented bythe formula III:

wherein

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or

wherein the bond between R₁ and N is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

or R₈ and R₉ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure; and

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In yet further embodiments, compounds include a halo-methyl ketoneanalog of an amino acid or amino acid derivative, such as athioxamide-modified amino acid. Exemplary compounds of this classinclude compounds represented by the formula IV:

wherein

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or

wherein the bond between R₁ and N is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

or R₈ and R₉ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure;

X₁, X₂ and X₃ each represent a hydrogen or a halogen; and

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain embodiments, compounds are peptides or peptidomimeticsincluding a prolyl group or analog thereof in the P1 specificityposition, and a nonpolar amino acid in the P2 specificity position,e.g., a nonpolar amino acid such as alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan or methionine, or an analogthereof, such as a thioxamide analog. For example, the compound mayinclude an Ala-Pro or Pro-Pro dipeptide sequence or equivalent thereof,and be represented in the formulas V and VI:

In certain embodiments, the ring A is a 5, 6 or 7 membered ring, e.g.,represented by

In certain embodiments, R₃₂ is a small hydrophobic group, e.g., a loweralkyl or a halogen.

In certain embodiments, R₃₀ represents a C-terminally linked amino acidresidue or amino acid analog, or a C-terminally linked peptide orpeptide analog, or an amino-protecting group wherein optionally whereapplicable the bond between R₃₀ and the N to which it is attached is athioxamide bond.

In certain embodiments, R₃₀ is H.

In certain embodiments, R₂ is absent, or is or a halogen, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

or small hydrophobic group such as a lower alkyl.

In certain embodiments, Z is C and R₃ is a hydrogen, or a smallhydrophobic group such as a lower alkyl or a halogen.

Another representative class of compounds for use in the subject methodinclude peptide and peptidomimetics of (D)-Ala-(L)-Ala, e.g., preservingthe diasteromeric orientation, in which one or more amide groups arereplaced by one or more thioxamide groups. Such compounds includecompounds represented by the formula VII:

wherein

W represents a functional group which reacts with an active site residueof the targeted protease, as for example, —CN, —CH═NR₅,

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

wherein optionally where applicable the bond between R₁ and the N towhich it is attached is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R′₇ represents, for each occurrence, hydrogen, or a substituted orunsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl,or heterocycle;

R₆₁ and R₆₂, independently, represent small hydrophobic groups;

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

In certain embodiments, R₁ is

wherein R₃₆ is a small hydrophobic group, e.g., a lower alkyl or ahalogen and R₃₈ is hydrogen, or, R₃₆ and R₃₈ together form a 4-7membered heterocycle including the N and the Cα carbon, as defined for Aabove; and R₄₀ represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group.

In certain embodiments, Z is C and R₃ is a hydrogen, or a smallhydrophobic group such as a lower alkyl or a halogen.

In certain embodiments, R₅ is a hydrogen, or a halogenated lower alkyl.

In certain embodiments, X₁ is a fluorine, and X₂ and X₃, if halogens,are fluorine.

In certain embodiments, R₆₁ and R₆₂, independently, represent lowalkyls, such as methyl, ethyl, propyl, isopropyl, tert-butyl or thelike.

Embodiment B

Another representative class of compounds for use in the method of thepresent invention are represented by formula VIII:

wherein

R₁ represents hydrogen, halogen or lower alkyl, lower alkenyl, or loweralkynyl, preferably lower alkyl such as methyl, ethyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₂ represents a branched lower alkyl, aralkyl, aryl, heteroaralkyl,heteroaryl, cycloalkyl, or cycloalkylalkyl, preferably a bulkyhydrophobic group, such as cyclohexyl, t-butyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₃ represents hydrogen or an amino-protecting group, preferablyhydrogen;

R₄ represents hydrogen, a C-terminally linked amino acid residue oramino acid analog, a C-terminally linked peptide or peptide analog, anamino-protecting group, or

preferably hydrogen, wherein optionally where applicable the bondbetween R₄ and the N to which it is attached is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇;

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R₁₁ and R₁₂ each independently represent hydrogen, an alkyl, or apharmaceutically acceptable salt, or R₁₁ and R₁₂ taken together with theO—B—O atoms to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure; and

m is zero or an integer in the range of 1 to 8.

In other embodiments, the subject compounds include aldehyde analogs ofalanine or alanyl derivatives, such as thioxamide-modified derivatives.Exemplary compounds of the present invention are represented by theformula IX:

wherein

R₁ represents hydrogen, halogen or lower alkyl, lower alkenyl, or loweralkynyl, preferably lower alkyl such as methyl, ethyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₂ represents a branched lower alkyl, aralkyl, aryl, heteroaralkyl,heteroaryl, cycloalkyl, or cycloalkylalkyl, preferably a bulkyhydrophobic group, such as cyclohexyl, t-butyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₃ represents hydrogen or an amino-protecting group, preferablyhydrogen;

R₄ represents hydrogen, a C-terminally linked amino acid residue oramino acid analog, a C-terminally linked peptide or peptide analog, anamino-protecting group, or

preferably hydrogen, wherein optionally where applicable the bondbetween R₄ and the N to which it is attached is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇;

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

m is zero or an integer in the range of 1 to 8.

In yet further embodiments, the compounds are halo-methyl ketone analogsof an amino acid or thioxamide-modified amino acid. Exemplary inhibitorsof this class include compounds represented by the formula X:

wherein

R₁ represents hydrogen, halogen or lower alkyl, lower alkenyl, or loweralkynyl, preferably lower alkyl such as methyl, ethyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₂ represents a branched lower alkyl, aralkyl, aryl, heteroaralkyl,heteroaryl, cycloalkyl, or cycloalkylalkyl, preferably a bulkyhydrophobic group, such as cyclohexyl, t-butyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₃ represents hydrogen or an amino-protecting group, preferablyhydrogen;

R₄ represents hydrogen, a C-terminally linked amino acid residue oramino acid analog, a C-terminally linked peptide or peptide analog, anamino-protecting group, or

preferably hydrogen, where optionally where applicable the bond betweenR₄ and the N to which it is attached is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇;

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

X₁, X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8.

In certain embodiments, the compound is a peptide or peptidomimeticincluding a alaninyl group or analog thereof, such as a thioxamideanalog, in the P1 specificity position, and a non-naturally occurringamino acid in the P2 specificity position, or an analog thereof, such asa thioxamide analog. For example, the compound may include anCyclohexylglycine-Ala or t-butylglycine-Ala dipeptide sequence orequivalent thereof, and be represented in the formula XI:

R₁ represents hydrogen, halogen or lower alkyl, lower alkenyl, or loweralkynyl, preferably lower alkyl such as methyl, ethyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₂ represents a branched lower alkyl, aralkyl, aryl, heteroaralkyl,heteroaryl, cycloalkyl, or cycloalkylalkyl, preferably a bulkyhydrophobic group, such as cyclohexyl, t-butyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₃ represents hydrogen or an amino-protecting group, preferablyhydrogen;

R₄ represents hydrogen, a C-terminally linked amino acid residue oramino acid analog, a C-terminally linked peptide or peptide analog, anamino-protecting group, or

preferably hydrogen, where optionally where applicable the bond betweenR₄ and the N to which it is attached is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇;

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

W represents a functional group which reacts with an active site residueof the targeted protease, as for example, —CN, —CH═NR₅₃,

preferably

Y₁ and Y₂ are, independently, OH, or a group capable of beinghydrolyzed, e.g., under physiologic conditions to a hydroxyl group, suchas alkoxy, aryloxy, etc., including cyclic derivatives where Y₁ and Y₂are connected via a ring having from 5 to 8 atoms in the ring structure(such as pinacol or the like);

R₅₀ represents O or S;

R₅₁ represents N₃, SH, NH₂, NO₂ or OR′₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

R₅₃ represents hydrogen, an alkyl, an alkenyl, an alkynyl,—C(X₁)(X₂)—X₃, —(CH₂)_(m)—R₇, —(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl,—(CH₂)_(n)—O-alkenyl, —(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇,—(CH₂)_(n)—SH, —(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl,—(CH₂)_(n)—S-alkynyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇, —C(O)C(O)NH₂,—C(O)C(O)OR′₇, preferably a hydrogen, or a halogenated lower alkyl;

X₁ represents a halogen, preferably a fluorine;

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

Another representative class of compounds for use in the subject methodinclude peptide and peptidomimetics of (L)-Ala-(L)-Cyclohexylglycine orthioxamide analogs thereof, e.g., preserving the steric disposition ofmoieties. Such inhibitors include compounds represented by the formulaXII:

wherein

R₁ represents hydrogen, halogen or lower alkyl, lower alkenyl, or loweralkynyl, preferably lower alkyl such as methyl, ethyl, etc., optionallysubstituted by one or more small substitutents such as halogen, hydroxy,alkoxy, etc.;

R₂ represents a branched lower alkyl, aralkyl, aryl, heteroaralkyl,heteroaryl, cycloalkyl, or cycloalkylalkyl, preferably a bulkyhydrophobic group, such as cyclohexyl, t-butyl, etc., optionallysubstituted by one or more small substituents such as halogen, hydroxy,alkoxy, etc.;

R₃ represents hydrogen or an amino-protecting group, preferablyhydrogen;

R₄ represents hydrogen, a C-terminally linked amino acid residue oramino acid analog, a C-terminally linked peptide or peptide analog, anamino-protecting group, or

preferably hydrogen, wherein optionally where applicable the bondbetween R₄ and the N to which it is attached is a thioxamide bond;

R₆ represents hydrogen, a halogen, alkyl, alkenyl, alkynyl, aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇;

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

W represents a functional group which reacts with an active site residueof the targeted protease, as for example, —CN, —CH═NR₅₃,

preferably

Y₁ and Y₂ are, independently, OH, or a group capable of beinghydrolyzed, e.g., under physiologic conditions to a hydroxyl group, suchas alkoxy, aryloxy, etc., including cyclic derivatives where Y₁ and Y₂are connected via a ring having from 5 to 8 atoms in the ring structure(such as pinacol or the like);

R₅₀ represents O or S;

R₅₁ represents N₃, SH, NH₂, NO₂ or OR′₇;

R₅₂ represents hydrogen, a lower alkyl, amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

R₅₃ represents hydrogen, alkyl, alkenyl, alkynyl, —C(X₁)(X₂)—X₃,—(CH₂)_(m)—R₇, —(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl, —(CH₂)_(n)—O-alkenyl,—(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(n)—SH,—(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇, —C(O)C(O)NH₂, —C(O)C(O)OR′₇, preferably ahydrogen, or a halogenated lower alkyl;

X₁ represents a halogen, preferably a fluorine;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

Embodiment C

A representative class of compounds for use in the method of the presentinvention are represented by formula XIII:

A-G  (XIII)

or a pharmaceutically acceptable salt thereof, wherein

A represents a peptidyl moiety which is a substrate for an activatingprotease;

A and G are covalently linked by a bond that is cleaved by theactivating protease;

G represents an inhibitor of a target protease which, when cleaved fromA by the activating serine protease, is characterized by one or both ofthe following: undergoes proto-deboronation and/or inhibits the targetprotease with a Ki of 100 nM or less; and

the compound of Formula XIII comprises one or more thioxamide groups.

In certain embodiments, the activating protease can be a serineprotease, a cysteine protease or a metalloprotease. Likewise, the targetprotease can be a serine protease, a cysteine protease or ametalloprotease. In certain embodiments, the target and activatingproteases are serine proteases.

In certain embodiments, the activating protease is a post-prolylcleaving protease, such as selected from the group consisting of DPP IV,DPP II, Prolyl oligopeptidase (PO), Fibroblast Activating Protein (FAP),and prolyl carboxypeptidase. In certain embodiments, the post-prolylcleaving protease is an endopeptidase, and A includes a blocked aminoterminus.

In other embodiments, the activating protease is selected from the groupconsisting of the group consisting of thrombin (Factor X), matriptase,falcipain, prostate specific antigen (PSA), and proteases homologousthereto.

In certain embodiments, the target protease is a post-prolyl cleavingprotease, such as selected from the group consisting of DPP IV, DPP II,Prolyl oligopeptidase (PO), Fibroblast Activating Protein (FAP), andprolyl carboxypeptidase.

In certain embodiments, G is a dipeptidyl moiety, e.g., derived fromnaturally occurring amino acids or analogs thereof.

In certain embodiments, G represents an inhibitor of a target proteasewhich, when cleaved from A by the activating serine protease, inhibitsthe target protease with a Ki of 100 nM or less, and certainembodiments, 10, 1 or 0.1 nM or less.

In certain embodiments, the half-life time (T_(1/2)) in serum for theinhibitor G is less than 24 hours, and even more preferably less than 10hours, 1 hour or even 10 min.

In certain embodiments, the address moiety A represents a C-terminallylinked peptide or peptide analog, e.g., of 2-10 amino acid residues,more preferably 2-4 residues, which is a substrate for the activatingenzyme. In certain embodiments, A is a dipeptidyl or tripepidyl moiety.In certain embodiments, A is derived from naturally occurring aminoacids or analogs thereof, and in certain embodiments, at least oneresidue of A is a non-naturally occurring amino acid analog.

In certain embodiments, such as when the address moiety A is a substrateof DPP IV, the amino terminus of the peptide or peptide analog isblocked with an amino-terminal protecting group, preferably a loweralkyl such as a methyl group.

In certain embodiments, the inhibitor moiety G is a dipeptidyl moietyand a electrophilic functional group that can form a covalent adductwith a residue in the active site of a protease replacing the carboxylterminus of the dipeptidyl moiety. For instance, the inhibitor moiety Gcan be represented in the formula XIV:

Xaa₁-Xaa₂-W  (XIV)

wherein

Xaa₁ is a naturally occurring amino acid or analog thereof, wherein Xaa₁contains a thioxamide group;

Xaa₂ is a naturally occurring amino acid or analog thereof;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

-   -   R₅ represents H, alkyl, alkenyl, alkynyl, —C(X₁)(X₂)X₃,        —(CH₂)_(m)—R₆, —(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl,        —(CH₂)_(n)—O-alkenyl, —(CH₂)_(n)—O-alkynyl,        —(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(n)—SH, —(CH₂)_(n)—S-alkyl,        —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,        —(CH₂)_(n)—S—(CH₂)_(m)—R₆, —C(O)C(O)NH₂, or —C(O)C(O)OR₇;

R₆ represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle;

R₇ represents independently for each occurrence hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and

Y₁ and Y₂ can independently or together be —OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

The pro-soft inhibitors of the present invention do not themselvesundergo proto-deboronation and can be constructed such that they do notinhibit the selected target enzyme, or other enzymes to any significantextent, before being cleaved by the activating protease. That is, thepro-soft inhibitors are themselves inactive, but produce an activeinhibitor moiety G in the body when the address moiety A is removed fromthe pro-soft inhibitor.

One of the features that makes the pro-soft inhibitor molecules of thecurrent invention different from typical prodrugs is that the inhibitormoiety, after being generated in the active form near the target,undergoes inactivation over time, e.g., as it diffuses away from thetarget enzyme, thereby reducing the possibility of deleterious sideeffects that may result from inhibition of enzymes occurring in otherparts of the patient. This combination of being released in an activeform in the vicinity of the target enzyme together with this“programmed” deactivation mechanism makes the molecules of the inventionmore specific, effective, and safer (i.e., having fewer side effects)than the inhibitor moiety used on its own.

In certain embodiments, the inhibitor moiety G is a dipeptidyl moiety,e.g., derived from naturally occurring amino acids or amino acid analogscomprising a thioamide moiety.

In certain embodiments, the inhibitor moiety G is an inhibitor of atarget protease which, when cleaved from pro-soft inhibitor by theactivating protease, inhibits the target protease with a K_(i) of 100 nM(10⁻⁷M) or less, and even more preferably, a Ki less than equal to 25nM, 10 nM (10⁻⁸M), 1 nM (10⁻⁹M), or 0.1 nM (10⁻¹⁰ M). In certainembodiments, K_(i)'s of less than 10⁻¹¹M and even 10⁻¹²M have beenmeasured or estimated for the subject inhibitor moieties.

In certain embodiments, the therapeutic index for the pro-soft inhibitoris at least 2 times greater than the therapeutic index for the inhibitormoiety alone, and even more preferably 5, 10, 50 or even 100 timesgreater.

For many of the subject pro-soft inhibitors, another improvement overthe inhibitor moiety itself is increased stability in pharmaceuticalpreparations, such as in solution, oils or solid formulations. Suchstability can be expressed in terms of shelf-life. In certain certainembodiments, the subject pro-soft inhibitor has a T₉₀ of at least 7days, and even more preferably of at least 20, 50, 100 or even 200 days.In certain embodiments, the subject pro-soft inhibitor has a T₅₀ of atleast 20 days, and even more preferably of at least 50, 100, 200 or even400 days. In certain embodiments, the subject pro-soft inhibitor has aT₉₀ as a solid, single oral dosage formulation of at least 20, 50, 100or even 200 days. In certain embodiments, the subject pro-soft inhibitorhas a T₉₀ as a liquid, single dosage suspension of at least 20, 50, 100or even 200 days.

Preferred pharmaceutical preparations of the subject pro-soft inhibitorsare substantially pyrogen-free. For example, in certain embodiments, theendotoxin concentration of the subject preparation, as assayed by thevia the gel-clot method (as a limits test with comparison to the maximumallowed FDA limit, as stated in appendix E of the Endotoxin Guidance),is less than 10 EU/mL or EU/single dosage formulation, and even morepreferably less than 5, 1, or even 0.1 EU/mL or EU/single dosageformulation.

In certain embodiments, a single administration of the pro-softinhibitor, such as bolus injection, oral dosage or inhaled dosage, canproduce a sustained in vivo effect, such as to provide a therapeuticallyeffective amount (≧ED₅₀ concentration) of the inhibitor moiety G for aperiod of at least 4 hours, and even more preferably at least 8, 12 oreven 16 hours.

In certain embodiments, the released inhibitor moiety G, andparticularly the inactive compound, has half-life (e.g., relative todecomposition into lower molecular weight fragments) in serum or otherbiologically relevant fluid of greater than 10 hours, and even morepreferably a half-life greater than 24, 48 or 120 hours.

Formulations of the present invention include those especiallyformulated for oral, buccal, parental, transdermal, inhalation,intranasal, transmucosal, implant, or rectal administration. In certainembodiments, the subject inhibitors are orally available, and can beprovided in the form of solid dosage formulations suitable for oraladministration to a human patient. In certain embodiments, the subjectinhibitors are transdermally active, and can be provided in the form oftopical cream or suspension or a transdermal patch.

Another aspect of the invention provides a pharmaceutical packageincluding one or more of the subject pro-soft inhibitors, andinstructions (written and/or pictorial) describing the administration ofthe formulation to a patient. Merely to illustrate, exemplary packagesare appropriately dosed and include instructions for one or more of:treatment or prophylaxis of metabolic disorders, gastrointestinaldisorders, viral disorders, inflammatory disorders, diabetes, obesity,hyperlipidemia, dermatological or mucous membrane disorders, psoriasis,intestinal distress, constipation, autoimmune disorders,encephalomyelitis, complement mediated disorders, glomerulonepritis,lipodystrophy; tissue damage, psychosomatic, depressive, andneuropsychiatric disorders, HIV infection, allergies, inflammation,arthritis, transplant rejection, high blood pressure, congestive heartfailure, tumors, and stress-induced abortions.

Preferably, the package includes the one or more pro-soft inhibitorsprovided as a single oral dosage formulation.

Where the pro-soft inhibitor includes one more chiral centers, incertain embodiments, the pro-soft inhibitor is provided as at least 75mol % of the eutomer (relative to the distomer) of that pro-softinhibitor, and even more preferably at least 85, 90, 95 or even 99 mol%. Generally, the eutomer with the L-enantiomer (with respect to the Cαcarbon) of an amino acid or amino acid analog.

In certain embodiments, the pro-soft inhibitor is a tetrapeptidyl moietyrepresented in the formula XV:

Xaa₁′-Xaa₂′-Xaa₁-Xaa₂-W  (XV)

wherein

Xaa₁′, Xaa₂′, and Xaa₂ each independently represent a naturallyoccurring amino acid or analog thereof;

Xaa₁ is a naturally occurring amino acid or analog thereof, wherein Xaa₁contains a thioxamide group;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

R₅ independently for each occurrence H, alkyl, alkenyl, alkynyl,—C(X₁)(X₂)X₃, —(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl,—(CH₂)n-O-alkenyl, —(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH,—(CH₂)n-S-alkyl, —(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl,—(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂, or —C(O)C(O)OR₇;

R₆ represents independently for each occurrence a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R₇ represents independently for each occurrence hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle;

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain embodiments, Xaa₁′ includes an amino-terminal protectinggroup.

In certain embodiments, Xaa₁′ is an amino acid analog having atetrasubstituted Cβ carbon, e.g., a carbon having four substituents noneof which is a hydrogen. For instance, Xaa₁′ can be an amino acid analogrepresented in the formula:

wherein: R₈ and R₉ each independently represent a lower alkyl or ahalogen; R₁₀ represents a lower alkyl, an aryl, a hydroxyl group or—(CH₂)_(m)—COOH; Z represents a hydrogen or an amino terminal protectinggroup; and m=0, 1 or 2. In certain embodiments, R₈ and R₉ eachindependently represents a lower alkyl, more preferably methyl, ethyl orpropyl, and even more preferably a methyl. In certain embodiments, R₁₀represents a lower alkyl, more preferably methyl, ethyl or propyl, andeven more preferably a methyl. In other certain embodiments, R₁₀represents an aryl, such as phenyl or hydroxyphenyl (preferablypara-hydroxy). In yet other certain embodiments, R₁₀ represents ahydroxyl group. In certain embodiments, R₁₀ represents —(CH₂)_(m)—COOH,where m=0, 1 or 2, and preferably where m is 0 or 1.

In certain embodiments, W is —B(Y₁)(Y₂).

In certain embodiments, R₂ is absent or represents halogen or loweralkyl.

In certain embodiments, R₄ represents hydrogen or lower alkyl.

In certain embodiments, R₅ represents H or alkyl.

In certain embodiments, Y₁ and Y₂ are OH.

In certain embodiments, W is —B(OH)₂.

In general, the subject pro-soft inhibitors can be divided into twodistinct types on the basis of whether they are activated by the same,or by a different enzyme as the target enzyme of the inhibitor moiety.The first type will be referred to as Type 1 or Target-Activated SmartProtease Inhibitors (TASPI), the second as Type 2 or Target-DirectedSmart Protease Inhibitors (TDSPI). Both embodiments of the pro-softinhibitors provide for the specific delivery of the active component tothe targeted enzyme and provide for attenuation of the inhibitoractivity as the inhibitor moiety diffuses away from the target enzyme.

TDSPIs of the present invention offer the additional prospects fortissue, or cellular specific inhibition of targeted enzymes. In otherwords TDSPIs offer the prospect of inhibiting a given enzyme in onegiven cell or tissue type but not in another. For example, every cell ofthe body contains a proteasome protease complex Inhibition of proteasomefunction has a number of practical therapeutic and prophylacticapplications. However, it is difficult to provide for inhibition ofproteasome activity in a cell- or tissue-type selective manner. Incertain embodiments of the current invention, TDSPIs can be constructedto deliver a proteasome inhibitor moiety in selective manner by using apro-soft inhibitor having an address moiety for a protease that isexpressed in or adjacent to the intended target cells or tissue. Toillustrate, it can be activated by FAP or Prostate Specific Antigen(PSA) and the resulting inhibitor moiety G is an inhibitor of theproteasome.

In certain embodiments of TDSPIs, the address moiety A is not anefficient substrate for the target protease. For instance, as asubstrate, address moiety A preferably has a turnover number as asubstrate for the target protease of less than 1/second, and even morepreferably less than 0.1/second, 0.001/second or even 0.0001/second.

In certain embodiments of the subject pro-soft inhibitors, the addressmoiety is a substrate for an activating protease selected from amongstserine proteases, cysteine proteases and metalloproteases. Likewise, theinhibitor moiety can be an dipeptidyl inhibitor for a target proteaseselected from serine proteases, cysteine proteases and metalloproteases.In certain embodiments, the target protease is a serine proteases.

The pro-soft inhibitors of the present invention can be designed to workwith target and activating serine proteases including, but not limitedto, dipeptidyl peptidase-11 (DPP-XI), dipeptidyl peptidase IV (DPP IV),dipeptidyl peptidase (DPP VIII), dipeptidyl peptidase 9 (DPP IX),aminopeptidase P, fibroblast activating protein alpha (seprase), prolyltripeptidyl peptidase, prolyl oligopeptidase (endoproteinase Pro-C),attractin (soluble dipeptidyl-aminopeptidase), acylaminoacyl-peptidase(N-acylpeptide hydrolase; fMet aminopeptidase) and lysosomal Pro-Xcarboxypeptidase (angiotensinase C, prolyl carboxypeptidase).

The pro-soft inhibitors of the present invention can be designed to workwith target and activating metalloproteases including membrane Pro-Xcarboxypeptidase (carboxypeptidase P), angiotensin-converting enzyme(Peptidyl-dipeptidase A multipeptidase], collagenase I (interstitialcollagenase; matrix metalloproteinase 1; MMP-1; Mcol-A), ADAM 10(alpha-secretase, myelin-associated disintegrin metalloproteinase),neprilysin (atriopeptidase; CALLA; CD10; endopeptidase 24.1 1;enkephalinase), Macrophage elastase (metalloelastase; matrixmetalloproteinase 12; MMP-12], Matrilysin (matrix metalloproteinase 7;MMP-7), and neurolysin (endopeptidase 24.16; microsomal endopeptidase;mitochondrial oligopeptidase).

In certain embodiments, the activating protease is a post-prolylcleaving protease, such as selected from the group consisting of DPP IV,DPP II, Prolyl oligopeptidase (PO), Fibroblast Activating Protein (FAP),and prolyl carboxypeptidase. In certain embodiments where thepost-prolyl cleaving protease is an endopeptidase, the amino terminus ofA is blocked with an amino-terminal protecting group, preferably a loweralkyl such as a methyl group.

In certain embodiments, the subject compound is represented by theformula XVI:

wherein

A represents a 4-8 membered heterocycle including the N and the Cαcarbon;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

R₁ represents a C-terminally linked peptide or peptide analog which is asubstrate for an activating enzyme;

R₂ is absent or represents one or more substitutions to the ring A, eachof which is independently a halogen, lower alkyl, lower alkenyl, loweralkynyl, carbonyl (such as a carboxyl, ester, formate, or ketone),thiocarbonyl (such as a thioester, thioacetate, or thioformate), amino,acylamino, amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R₇,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R₇, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

R₃ represents a hydrogen or a substituent which does not conjugate theelectron pair of the nitrogen to which it is attached, such as a loweralkyl;

R₄ represents hydrogen, halogen, a lower alkyl, lower alkenyl, loweralkynyl, aryl, or aralkyl;

R₅ represents H, alkyl, alkenyl, alkynyl, —C(X₁)(X₂)X₃, —(CH₂)_(m)—R₆,—(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl, —(CH₂)_(n)—O-alkenyl,—(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(n)—SH,—(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₆, —C(O)C(O)NH₂, or —C(O)C(O)OR₇;

R₆ represents independently for each occurrence a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R₇ represents independently for each occurrence hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle;

R₈ represents hydrogen, —CH₃, or —(CH₂)_(n)—CH₃;

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like);

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain embodiments, R₂ is absent, or represents a small hydrophobicgroup.

In certain embodiments, A represents a 5-membered heterocycle includingthe N and the Cα carbon.

In certain embodiments, W is —B(Y₁)(Y₂).

In certain embodiments, R₂ is absent or represents halogen or loweralkyl.

In certain embodiments, R₄ represents hydrogen or lower alkyl.

In certain embodiments, R₅ represents H or alkyl.

In certain embodiments, Y₁ and Y₂ are OH.

In certain embodiments, W is —B(OH)₂.

In certain embodiments, W is —B(OH)₂, and A represents a 5-memberedheterocycle including the N and the Cα carbon.

In certain embodiments, W is —B(OH)₂, A represents a 5-memberedheterocycle including the N and the Cα carbon, and R₂ is absent orrepresents halogen or lower alkyl.

In certain embodiments, W is —B(OH)₂, A represents a 5-memberedheterocycle including the N and the Cα carbon, R₂ is absent orrepresents halogen or lower alkyl, and

R₄ represents hydrogen or lower alkyl.

In other embodiments, W is —B(OH)₂, A represents a 5-memberedheterocycle including the N and the Cα carbon, R₄ represents hydrogen orlower alkyl, and R₂ is azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

In certain embodiments, R₁ is one of the following:

In certain embodiments, W is —B(OH)₂, A represents a 5-memberedheterocycle including the N and the Cα carbon, R₂ is absent orrepresents halogen or lower alkyl, and R₁ is one of the following:

In other embodiments, W is —B(OH)₂, A represents a 5-memberedheterocycle including the N and the Cα carbon, R₄ represents hydrogen orlower alkyl, and R₂ is azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

and R₁ is one of the following:

In certain embodiments, the compound is represented in the formula XVII:

wherein R₁, R₃, R₄ and W are as defined above, and p is an integer from1 to 3. In certain certain embodiments, p is 1, and R₃ is a hydrogen ineach occurrence.

In certain embodiments of the compound structures above, W represents:

In certain embodiments of the compound structures above, R₅ is ahydrogen or —C(X₁)(X₂)X₃, wherein X₁ is a fluorine, and X₂ and X₃, ifhalogens, are also fluorine.

In certain embodiments of the compound structures above, R₄ is a loweralkyl. In certain embodiments of the compound structures above, R₄represents a side chain of an amino acid residue selected from Gly, Ala,Val, Ser, Thr, Ile and Leu.

In certain embodiments of the compound structures above, R₁ is apeptidyl moiety which is a substrate for a post-proline cleaving enzyme.

In certain embodiments of the subject pro-soft inhibitor structures XVIand XVII, R₄ represents a side chain of an amino acid residuerepresented in the formula:

wherein

R_(4a) and R_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen;

R_(4c) represents a halogen, an amine, an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano; and

z is zero or an integer in the range of 0 to 3.

In certain embodiments of structures XIV and XV, R₄ represents a sidechain of an amino acid residue represented in the formula:

wherein: R₈ and R₉ each independently represent a lower alkyl or ahalogen; R₁₀ represents a lower alkyl, an aryl, a hydroxyl group or—(CH₂)_(m)—COOH. In certain embodiments, R₈ and R₉ each independentlyrepresents a lower alkyl, more preferably methyl, ethyl or propyl, andeven more preferably a methyl. In certain embodiments, R₁₀ represents alower alkyl, more preferably methyl, ethyl or propyl, and even morepreferably a methyl. In other certain embodiments, R₁₀ represents anaryl, such as phenyl or hydroxyphenyl (preferably para-hydroxy). In yetother certain embodiments, R₁₀ represents a hydroxyl group. In certainembodiments, R₁₀ represents —(CH₂)_(m)—COOH, where m=0, 1 or 2, andpreferably where m is 0 or 1.

In certain embodiments, the pro-soft inhibitor is activated by oneprotease and inhibits a different protease. For example, it can beactivated by FAP and the resulting inhibitor G is selective for theproteasome. In certain embodiments of the compound structures above, R₁is not an efficient substrate for the target protease. For instance, asa substrate, R₁ preferably has a turnover number as a substrate for thetarget protease of less than 1/second, and even more preferably lessthan 0.1/second, 0.001/second or even 0.0001/second.

In certain embodiments, A is a peptidyl moiety of 2 to 5 amino acidresidues or the equivalents thereof. In certain embodiments, A is adipeptidyl moiety, e.g., derived from naturally occurring amino acids oranalogs thereof.

In certain embodiments, the backbone of the peptidyl moiety A caninclude one or more be a non-hydrolyzable analogs of a peptide bond,except for the bond linking A to G.

In certain embodiments, A is represented by:

In certain embodiments of the present invention, compounds representedby the formula: A-G do not inhibit the selected target enzyme, or otherenzymes to an appreciable extent. In certain embodiments of the presentinvention, the pro-soft inhibitor are themselves inactive, but becomeactivated in the body when the R-A group is removed to liberate theenzyme inhibitory moiety G.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R₁ is one of the following:

Embodiment D

A representative class of compounds for use in the method of the presentinvention are represented by formula XVIII:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ represents H, alkyl, alkoxy, alkenyl, alkynyl, amino, alkylamino,acylamino, cyano, sulfonylamino, acyloxy, aryl, cycloalkyl,heterocyclyl, heteroaryl, or a polypeptide chain of 1 to 8 amino acidresidues;

R² and R³ each independently represent H, lower alkyl, and aralkyl, orR² and R³ together with the atoms to which they are attached, form a 4-to 6-membered heterocyclic ring;

R⁴ and R⁵ each independently represent H, halogen, or alkyl, or R⁴ andR⁵, together with the carbon to which they are attached, form a 3- to6-membered carbocyclic or heterocyclic ring;

R⁶ represents a functional group that reacts with an active site residueof a targeted protease to form a covalent adduct;

R⁷ is absent or represents one or more substituents on ring A, each ofwhich is independently selected from H, lower alkyl, lower alkenyl,lower alkynyl, hydroxyl, oxo, ether, thioether, halogen, carbonyl,thiocarbonyl, amino, amido, cyano, nitro, azido, alkylamino, acylamino,aminoacyl, cyano, sulfate, sulfonate, sulfonyl, sulfonylamino,aminosulfonyl, alkoxycarbonyl, acyloxy, aryl, cycloalkyl, heterocyclyl,heteroaryl, or polypeptide chains of 1 to 8 amino acid residues;

R⁸ represents H, aryl, alkyl, aralkyl, cycloalkyl, heterocyclyl,heteroaryl, heteroaralkyl, or a polypeptide chain of 1 to 8 amino acidresidues;

L is absent or represents alkyl, alkenyl, alkynyl,—(CH₂)_(m)—O—(CH₂)_(m)—, —(CH₂)_(m)NR₂(CH₂)_(m)—, or—(CH₂)_(m)S(CH₂)_(m)—;

X is absent or represents —N(R⁸)—, —O—, or —S—;

Y is absent or represents —C(═O)—, —C(═S)—, or —SO₂—;

m is, independently for each occurrence, an integer from 0 to 10; and

n is an integer from 0 to 3, preferably 0 or 1.

In certain embodiments, R¹ represents H or lower alkyl, R² and R³ eachindependently represent H, lower alkyl, or aralkyl, or R² and R³together with the atoms to which they are attached, form a 5-memberedheterocyclic ring, R⁴ represents H or lower alkyl, and R⁵ represents H.

In a further certain embodiment, the stereochemical designations at C3and C4 are R and S respectively.

In certain other embodiments, R⁶ represents cyano, boronic acid, —SO₂Z¹,—P(═O)Z¹, —P(R⁹)R¹⁰R¹¹, —C(═NH)NH₂, —CH═NR¹², or —C(═O)—R¹², wherein:

R⁹ represents O or S;

R¹⁰ represents N₃, SH₂, NH₂, NO₂, or OLR¹³, and

R¹¹ represents lower alkyl, amino, OLR¹³, or a pharmaceuticallyacceptable salt thereof, or

R¹⁰ and R¹¹, together with the phosphorus to which they are attached,form a 5- to 8-membered heterocyclic ring;

R¹² represents H, alkyl, alkenyl, alkynyl, —(CH₂)_(p)—R¹³,—(CH₂)_(q)—OH, —(CH₂)_(q)—O-alkyl, —(CH₂)_(q)—O-alkenyl,—(CH₂)_(q)—O-alkynyl, —(CH₂)_(q)—O—(CH₂)_(p)—R¹³, —(CH₂)_(q)—SH,—(CH₂)_(q)—S-alkyl, —(CH₂)_(q)—S-alkenyl, —(CH₂)_(q)—S-alkynyl,—(CH₂)_(q)—S—(CH₂)_(p)—R¹³, —C(O)C(O)NH₂, —C(O)C(O)OR¹⁴, or—C(Z¹)(Z²)(Z³);

R¹³ represents H, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, orheterocyclyl;

R¹⁴ represents H, alkyl, alkenyl, or LR¹³;

Z¹ represents a halogen;

Z² and Z³ independently represent H or halogen;

p is, independently for each occurrence, an integer from 0 to 8; and

q is, independently for each occurrence, an integer from 1 to 8.

In another embodiment, R⁶ represents CN, CHO, or C(═O)C(Z¹)(Z²)(Z³),wherein Z¹ represents a halogen, and Z² and Z³ represent H or halogen.In certain such embodiments, R⁶ represents C(═O)C(Z¹)(Z²)(Z³), whereinZ¹ represents fluorine, and Z² and Z³ represent H or fluorine.

In certain embodiments, R⁶ is a group represented by B(Y¹)(Y²) whereinY¹ and Y² are independently OH or a group that is hydrolysable to OH(i.e., to form a boronic acid), or together with the boron atom to whichthey are attached form a 5- to 8-membered ring that is hydrolysable to aboronic acid.

Embodiment E

A representative class of compounds for use in the method of the presentinvention are represented by formula XIX:

wherein

R¹ represents H, alkyl, alkoxy, alkenyl, alkynyl, amino, alkylamino,acylamino, cyano, sulfonylamino, acyloxy, aryl, cycloalkyl,heterocyclyl, heteroaryl, or a polypeptide chain of 1 to 8 amino acidresidues;

R² represents H, lower alkyl, or aralkyl;

R³ and R⁴ independently represent H, halogen, or alkyl, or R³ and R⁴together with the atoms to which they are attached, form a 3- to6-membered heterocyclic ring;

R⁵ represents H, halogen, lower alkyl, or aralkyl;

R⁶ represents a functional group that reacts with an active site residueof a targeted protease to form a covalent adduct;

R⁷ represents H, aryl, alkyl, aralkyl, cycloalkyl, heterocyclyl,heteroaryl, heteroaralkyl, or polypeptide chains of 1 to 8 amino acidresidues;

L is absent or represents alkyl, alkenyl, alkynyl,—(CH₂)_(m)O(CH₂)_(m)—, —(CH₂)_(m)NR₂(CH₂)_(m)—, and—(CH₂)_(m)S(CH₂)_(m)—;

X is absent or represents —N(R⁷)—, —O—, or —S—;

Y is absent or represents —C(═O)—, —C(═S)—, or —SO₂—;

m is, independently for each occurrence, an integer from 0 to 10; and

n is an integer from 1 to 6.

In certain embodiments, R¹ represents H or lower alkyl, R³ and R⁴together with the atoms to which they are attached form a 5-memberedring, and n is 2.

In certain other certain embodiments R¹ represents H or lower alkyl, R³represents H, R⁴ represents H or lower alkyl, R⁵ represents H, and n is2.

In certain embodiments, R¹ is a polypeptide chain of 2 to 8 amino acidresidues, wherein proline is the residue that is directly attached. Mostpreferably R¹ is a polypeptide chain of 2 amino acid residues

In certain above embodiments, R⁶ represents cyano, boronic acid, —SO₂Z¹,—P(═O)Z¹, —P(R⁸)R⁹R¹⁰, —C(═NH)NH₂, —CH═NR¹¹, and —C(═O)—R¹¹, wherein

R⁸ represents O or S;

R⁹ represents N₃, SH₂, NH₂, NO₂, and OLR¹², and

R¹⁰ represents lower alkyl, amino, OLR¹², or a pharmaceuticallyacceptable salt thereof, or

R⁹ and R¹⁰, together with the phosphorus to which they are attached,form a 5- to 8-membered heterocyclic ring;

R¹¹ represents H, alkyl, alkenyl, alkynyl, —(CH₂)_(p)—R¹²,—(CH₂)_(q)—OH, —(CH₂)_(q)—O-alkyl, —(CH₂)_(q)—O-alkenyl,—(CH₂)_(q)—O-alkynyl, —(CH₂)_(q)—O—(CH₂)_(p)—R¹², —(CH₂)_(q)—SH,—(CH₂)_(q)—S-alkyl, —(CH₂)_(q)—S-alkenyl, —(CH₂)_(q)—S-alkynyl,—(CH₂)_(q)—S—(CH₂)_(p)—R¹², —C(O)C(O)NH₂, —C(O)C(O)OR¹³, or—C(Z¹)(Z²)(Z³);

R¹² represents H, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, andheterocyclyl;

R¹³ represents H, alkyl, alkenyl, and LR¹²;

Z¹ represents a halogen;

Z² and Z³ independently represent H or halogen;

p is, independently for each occurrence, an integer from 0 to 8; and

q is, independently for each occurrence, an integer from 1 to 8.

In another embodiment, R⁶ represents CN, CHO, or C(═O)C(Z¹)(Z²)(Z³),wherein Z¹ represents a halogen, and Z² and Z³ represent H or halogen.In certain such embodiments, R⁶ represents C(═O)C(Z¹)(Z²)(Z³), whereinZ¹ represents fluorine, and Z² and Z³ represent H or fluorine.

In certain embodiments, R⁶ represents a group —B(Y¹)(Y²), wherein Y¹ andY² are independently OH or a group that is hydrolysable to OH (i.e.,thereby forming a boronic acid), or together with the boron atom towhich they are attached form a 5- to 8-membered ring that ishydrolysable to a boronic acid.

In certain embodiments, R³ and R⁴ together with the atoms to which theyare attached form a 5-membered ring, which is substituted with one ormore groups selected from hydroxyl, lower alkyl (e.g., methyl), loweralkenyl, lower alkynyl, lower alkoxy, lower hydroxyalkyl (e.g.,hydroxymethyl), and lower alkoxyalkyl.

In more certain embodiments, the substituent group is selected from thegroup consisting of lower alkyl, lower hydroxyalkyl and loweralkoxyalkyl. In more preferred such embodiments, the substituent groupis located at the 5-position of the ring.

In other more certain embodiments, the substituent group is hydroxyl,which is preferably located at the 4-position of the ring.

In certain embodiments, the substituent group on the 5-membered ringcontaining R³ and R⁴ is selected from the group consisting of loweralkyl (e.g., methyl), hydroxyl, lower hydroxyalkyl (e.g., hydroxymethyl)and lower alkoxyalkyl. In certain preferred such embodiments, thesubstituent group has a cis-stereochemical relationship to R⁶. Suchstereochemical relationships are particularly advantageous for compoundshaving substituents at the 4- or 5-position of the 5-membered ring, asdiscussed immediately above.

In certain embodiments of the invention, a subject compound has astructure of Formula XX:

or a pharmaceutically acceptable salt thereof, where:

R¹ represents H, alkyl, alkoxy, alkenyl, alkynyl, amino, alkylamino,acylamino, cyano, sulfonylamino, acyloxy, aryl, cycloalkyl,heterocyclyl, heteroaryl, or a polypeptide chain of 1 to 8 amino acidresidues;

R² represents H, lower alkyl, or aralkyl;

R³ and R⁴ independently represent H, halogen, or alkyl, or R³ and R⁴together with the carbon to which they are attached, form a 3- to6-membered heterocyclic ring;

R⁵ represents H, halogen, lower alkyl, or aralkyl, preferably H or loweralkyl;

R⁶ represents a functional group that reacts with an active site residueof the targeted protease to form a covalent adduct;

R⁷ represents H, aryl, alkyl, aralkyl, cycloalkyl, heterocyclyl,heteroaryl, heteroaralkyl, or a polypeptide chain of 1 to 8 amino acidresidues;

R¹⁴ represents H, alkyl, alkoxy, alkenyl, alkynyl, or aralkyl,preferably H;

A is absent or represents —NHC(═NH)—, or R¹⁴ and A together with thenitrogen to which they are attached form a heterocyclic ring;

L is absent or represents alkyl, alkenyl, alkynyl, (CH₂)_(m)O(CH₂)_(m)—,—(CH₂)_(m)NR₂(CH₂)_(m)—, and —(CH₂)_(m)S(CH₂)_(m)—;

X is absent or represents —N(R⁷)—, —O—, or —S—;

Y is absent or represents —C(═O)—, —C(═S)—, or —SO₂—;

m is, independently for each occurrence, an integer from 0 to 10; and

n is an integer from 1 to 6.

In certain embodiments, R¹ represents H or lower alkyl, R³ and R⁴together with the carbon to which they are attached form a 5-memberedring, and n is an integer from 1 to 4.

In certain embodiments, R¹⁴ is H, A is absent, and n is 4. In certainother embodiments, R¹⁴ is H, A is —NHC(═NH)—, and n is 3.

In certain embodiments, A and R¹⁴ together with the nitrogen to whichthey are attached form an imidazole ring, and n is 1.

In certain embodiments, R⁶ represents boronic acid, CN, —SO₂Z¹,—P(═O)Z¹, —P(═R⁸)R⁹R¹⁰, —C(═NH)NH₂, —CH═NR¹¹, or —C(═O)—R¹¹ wherein

R⁸ is O or S;

R⁹ represents N₃, SH₂, NH₂, NO₂, or OLR¹², and

R¹⁰ represents lower alkyl, amino, OLR¹², or a pharmaceuticallyacceptable salt thereof, or

R⁹ and R¹⁰, together with the phosphorus to which they are attached,form a 5- to 8-membered heterocyclic ring;

R¹¹ represents H, alkyl, alkenyl, alkynyl, NH₂, —(CH₂)_(p)—R¹²,—(CH₂)_(q)—OH, —(CH₂)_(q)—O-alkyl, —(CH₂)_(q)—O-alkenyl,—(CH₂)_(q)—O-alkynyl, —(CH₂)_(q)—O—(CH₂)_(p)—R¹², —(CH₂)_(q)—SH,—(CH₂)_(q)—S-alkyl, —(CH₂)_(q)—S-alkenyl, —(CH₂)_(q)—S-alkynyl,—(CH₂)_(q)—S—(CH₂)_(p)—R¹², —C(O)NH₂, —C(O)OR¹³, or C(Z¹)(Z²)(Z³);

R¹² represents H, alkyl, alkenyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, or heterocyclyl;

R¹³ represents H, alkyl, alkenyl, or LR¹²;

Z¹ represents a halogen;

Z² and Z³ independently represent H or halogen;

p is, independently for each occurrence, an integer from 0 to 8; and

q is, independently for each occurrence, an integer from 1 to 8.

In certain embodiments, R⁶ represents CN, CHO, or C(═O)C(Z¹)(Z²)(Z³),wherein Z¹ represents a halogen, and Z² and Z³ represent H or halogen.In another embodiment, R⁶ represents C(═O)C(Z¹)(Z²)(Z³), wherein Z¹represents fluorine, and Z² and Z³ represent H or fluorine.

In certain embodiments, R⁶ represents a group —B(Y¹)(Y²), wherein Y¹ andY² are independently OH or a group that is hydrolysable to OH, ortogether with the boron atom to which they are attached form a 5- to8-membered ring that is hydrolysable to a boronic acid.

In certain embodiments, R³ and R⁴ together with the atoms to which theyare attached form a 5-membered ring, which is substituted with one ormore groups selected from hydroxyl, lower alkyl (e.g., methyl), loweralkenyl, lower alkynyl, lower alkoxy, lower hydroxyalkyl (e.g.,hydroxymethyl), and lower alkoxyalkyl.

In more certain embodiments, the substituent group is selected from thegroup consisting of lower alkyl, lower hydroxyalkyl and loweralkoxyalkyl. In more preferred such embodiments, the substituent groupis located at the 5-position of the ring.

In other more certain embodiments, the substituent group is hydroxyl,which is preferably located at the 4-position of the ring.

In certain embodiments, the substituent group on the 5-membered ringcontaining R³ and R⁴ is selected from the group consisting of loweralkyl (e.g., methyl), hydroxyl, lower hydroxyalkyl (e.g., hydroxymethyl)and lower alkoxyalkyl. In certain preferred such embodiments, thesubstituent group has a cis-stereochemical relationship to R⁶. Suchstereochemical relationships are particularly advantageous for compoundshaving substituents at the 4- or 5-position of the 5-membered ring, asdiscussed immediately above.

In certain embodiments of the invention, a subject compound has astructure of Formula XXI:

or a pharmaceutically acceptable salt thereof, where:

R¹ represents H, alkyl, alkoxy, alkenyl, alkynyl, amino, alkylamino,acylamino, cyano, sulfonylamino, acyloxy, aryl, cycloalkyl,heterocyclyl, heteroaryl, or a polypeptide chain of 1 to 8 amino acidresidues;

R² represents H, lower alkyl, or aralkyl;

R³ and R⁴ independently represent H, halogen, or alkyl, or R³ and R⁴together with the carbon to which they are attached, form a 3- to6-membered heterocyclic ring;

R⁵ represents H, halogen, lower alkyl, or aralkyl, preferably H or loweralkyl;

R⁶ represents a functional group that reacts with an active site residueof a targeted protease to form a covalent adduct;

R⁷ represents H, aryl, alkyl, aralkyl, cycloalkyl, heterocyclyl,heteroaryl, heteroaralkyl, or a polypeptide chain of 1 to 8 amino acidresidues;

R¹⁵ is a functional group that has either a positive or negative chargeat physiological pH, preferably an amine or carboxylic acid;

L is absent or represents alkyl, alkenyl, alkynyl,—(CH₂)_(m)O(CH₂)_(m)—, —(CH₂)_(m)NR₂(CH₂)_(m)—, and—(CH₂)_(m)S(CH₂)_(m)—;

X is absent or represents —N(R⁷)—, —O—, or —S—;

Y is absent or represents —C(═O)—, —C(═S)—, or —SO₂—;

m is, independently for each occurrence, an integer from 0 to 10; and

n is an integer from 1 to 6.

In certain embodiments, R¹ represents H or lower alkyl, R³ is H and R⁴is lower alkyl, or R³ and R⁴ together with the carbon to which they areattached form a 5-membered ring, and n is an integer from 1 to 4.

In certain embodiments, n is an integer from 1 to 4 and R¹⁵ is afunctional group that has either a positive or negative charge atphysiological pH. In more certain embodiments n is an integer from 1 to4 and R¹⁵ is selected from the group consisting of amine, carboxylicacid, imidazole, or guanidine functionality.

In certain embodiments, R⁶ represents boronic acid, CN, —SO₂Z¹,—P(═O)Z¹, —P(═R⁸)R⁹R¹⁰, —C(═NH)NH₂, —CH═NR¹¹, or —C(═O)—R¹¹ wherein

R⁸ is O or S;

R⁹ represents N₃, SH₂, NH₂, NO₂, or OLR¹², and

R¹⁰ represents lower alkyl, amino, OLR¹², or a pharmaceuticallyacceptable salt thereof, or

R⁹ and R¹⁰, together with the phosphorus to which they are attached,form a 5- to 8-membered heterocyclic ring;

R¹¹ represents H, alkyl, alkenyl, alkynyl, NH₂, —(CH₂)_(p)—R¹²,—(CH₂)_(q)—OH, —(CH₂)_(q)—O-alkyl, —(CH₂)_(q)—O-alkenyl,—(CH₂)_(q)—O-alkynyl, —(CH₂)_(q)—O—(CH₂)_(p)—R¹², —(CH₂)_(q)—SH,—(CH₂)_(q)—S-alkyl, —(CH₂)_(q)—S-alkenyl, —(CH₂)_(q)—S-alkynyl,—(CH₂)_(q)—S—(CH₂)_(p)—R¹², —C(O)NH₂, —C(O)OR¹³, or —C(Z¹)(Z²)(Z³);

R¹² represents H, alkyl, alkenyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, or heterocyclyl;

R¹³ represents H, alkyl, alkenyl, or LR¹²;

Z¹ represents a halogen;

Z² and Z³ independently represent H or halogen;

p is, independently for each occurrence, an integer from 0 to 8; and

q is, independently for each occurrence, an integer from 1 to 8.

In certain embodiment, R⁶ represents CN, CHO, or C(═O)C(Z¹)(Z²)(Z³),wherein Z¹ represents fluorine, and Z² and Z³ represent H or fluorine.In another embodiment, R⁶ represents C(═O)C(Z¹)(Z²)(Z³), wherein Z¹represents fluorine, and Z² and Z³ represent H or fluorine.

In certain embodiments, R⁶ represents a group —B(Y¹)(Y²), wherein Y¹ andY² are independently OH or a group that is hydrolysable to OH, ortogether with the boron atom to which they are attached form a 5- to8-membered ring that is hydrolysable to a boronic acid.

In certain embodiments, R³ and R⁴ together with the atoms to which theyare attached form a 5-membered ring substituted with one or more groupsselected from hydroxyl, lower alkyl (e.g., methyl), lower alkenyl, loweralkynyl, lower alkoxy, lower hydroxyalkyl (e.g., hydroxymethyl), andlower alkoxyalkyl.

In more certain embodiments, the substituent group is selected from thegroup consisting of lower alkyl, lower hydroxyalkyl and loweralkoxyalkyl. In more preferred such embodiments, the substituent groupis located at the 5-position of the ring.

In other more certain embodiments, the substituent group is hydroxyl,which is preferably located at the 4-position of the ring.

In certain embodiments, the substituent group on the 5-membered ringcontaining R³ and R⁴ is selected from the group consisting of loweralkyl (e.g., methyl), hydroxyl, lower hydroxyalkyl (e.g., hydroxymethyl)and lower alkoxyalkyl. In certain preferred such embodiments, thesubstituent group has a cis-stereochemical relationship to R⁶. Suchstereochemical relationships are particularly advantageous for compoundshaving substituents at the 4- or 5-position of the 5-membered ring, asdiscussed immediately above.

Another aspect of the invention relates to inhibitors having a structureof Formula XXII:

or a pharmaceutically acceptable salt thereof, wherein

A is selected from the group consisting of a 4-8 membered heterocycleincluding the N and a Cα carbon;

Z is C or N;

W is selected from the group consisting of CN, —CH═NR⁵, a functionalgroup which reacts with an active site residue of the targeted protease,

R¹ is selected from the group consisting of a C-terminally linked aminoacid residue or amino acid analog, a C-terminally linked peptide orpeptide analog, or

wherein the bond between R¹ and N is a thioxamide bond;

R² represents one or more substitutions to the ring A, each of which isindependently a halogen, lower alkyl, lower alkenyl, lower alkynyl,carbonyl, carboxyl, ester, formate, ketone, thiocarbonyl, thioester,thioacetate, thioformate, amino, acylamino, amido, nitro, sulfate,sulfonate, sulfonamido, —(CH₂)_(m)—R⁷, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-loweralkyl, —(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R⁷,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl, or—(CH₂)_(n)—S—(CH₂)_(m)—R⁷, azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

wherein at least one R² is selected from the group consisting of —OH,lower alkyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl,preferably at least one of lower alkyl (e.g., methyl), lower alkoxy,lower hydroxyalkyl (e.g., hydroxymethyl), and lower alkoxyalkyl;

when Z is N, R³ is absent;

when Z is C, R³ is selected from the group consisting of hydrogen,halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl,thiocarbonyl, amino, acylamino, amido, cyano, nitro, azido, sulfate,sulfonate, sulfonamido, —(CH₂)_(m)—R⁷, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-loweralkyl, —(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R⁷,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl, and—(CH₂)_(n)—S—(CH₂)_(m)—R⁷;

R⁵ is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, —C(X¹)(X²)X³, —(CH₂)_(m)—R⁷, —(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl,—(CH₂)_(n)—O-alkenyl, —(CH₂)_(n)—O—-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R⁷,—(CH₂)_(n)—SH, —(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl,—(CH₂)_(n)—S-alkynyl, —(CH₂)_(n)—S—(CH₂)_(m)—R⁷, —C(O)C(O)NH₂, and—C(O)C(O)OR^(7′);

R⁶ is selected from the group consisting of hydrogen, halogen, alkyl,alkenyl, alkynyl, aryl, —(CH₂)_(m)—R⁷, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl,—(CH₂)_(m)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl,—(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R⁷,

each R⁷ is independently selected from aryl, aralkyl, cycloalkyl,cycloalkenyl, and heterocyclyl;

each R^(7′) is independently selected from hydrogen, alkyl, alkenyl,aryl, aralkyl, cycloalkyl, cycloalkenyl and heterocyclyl;

R⁸ and R⁹ are each independently selected from hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R⁷, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl, and—C(═O)—(CH₂)_(m)—R⁷; or

R⁸ and R⁹ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure;

R¹⁰ represents hydrogen, —CH₃, or —(CH₂)_(n)—CH₃;

R⁵⁰ is O or S;

R⁵¹ is selected from the group consisting of N₃, SH, NH₂, NO₂, andOR^(7′);

R⁵² is selected from the group consisting of hydrogen, lower alkyl,amine, OR^(7′), or a pharmaceutically acceptable salt thereof, or

R⁵¹ and R⁵² taken together with the P atom to which they are attachedcomplete a heterocyclic ring having from 5 to 8 atoms in the ringstructure;

X¹ is a halogen;

X² and X³ are each selected from hydrogen and halogen;

Y¹ and Y² are each independently selected from OH and a group capable ofbeing hydrolyzed to OH, including cyclic derivatives where Y¹ and Y² areconnected via a ring having from 5 to 8 atoms in the ring structure;

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

In certain embodiments, W is selected from the group consisting of CNand B(Y¹)(Y²). In certain embodiments, A is a five-membered ring, Z isC, and W is B(Y¹)(Y²).) In more certain embodiments, Z has the absolutestereochemical configuration of L-proline.

In certain embodiments, A is a five-membered ring, Z is C, and R² isselected from the group consisting of hydroxyl, lower alkyl, loweralkenyl, lower alkynyl, lower alkoxy, lower hydroxyalkyl, and loweralkoxyalkyl. In certain preferred such embodiments, R² is selected fromthe group consisting of lower hydroxyalkyl and lower alkoxyalkyl. Inmore preferred such embodiments, R² is located at the 5-position of thering.

In certain embodiments, A is a five-membered ring, Z is C, and R² isselected from the group consisting of hydroxyl, lower alkyl (such asmethyl), lower hydroxyalkyl (such as hydroxymethyl) and loweralkoxyalkyl. In certain preferred such embodiments, Z has the absolutestereochemical configuration of L-proline and R² is located at the5-position of the ring for lower alkyl, lower hydroxyalkyl and loweralkoxyalkyl and at the 4-position for hydroxyl. In more preferred suchembodiments, R² has a cis-stereochemical relationship to W.

Another aspect of the invention relates to inhibitors having a structureof Formula XXIII:

or a pharmaceutically acceptable salt thereof, wherein

R¹ is selected from the group consisting of a C-terminally linked aminoacid residue or amino acid analog, a C-terminally linked peptide orpeptide analog, or

wherein the bond between R¹ and N is a thioxamide bond;

R² represents one or more substitutions to the ring A, each of which isindependently a halogen, lower alkyl, lower alkenyl, lower alkynyl,carbonyl (such as a carboxyl, ester, formate, or ketone), thiocarbonyl(such as a thioester, thioacetate, or thioformate), amino, acylamino,amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R⁷,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R⁷, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

wherein at least one R² is selected from the group consisting of —OH,lower alkyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl,preferably at least one of lower alkyl (e.g., methyl), lower alkoxy,lower hydroxyalkyl (e.g., hydroxymethyl), and lower alkoxyalkyl;

R⁶ is selected from the group consisting of hydrogen, halogen, alkyl,alkenyl, alkynyl, aryl, —(CH₂)_(m)—R⁷, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl,—(CH₂)_(m)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl,—(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R⁷,

R⁷ is selected from the group consisting of aryl, cycloalkyl,cycloalkenyl, and heterocyclyl;

R⁸ and R⁹ are each independently selected from hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R⁷, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl, and—C(═O)—(CH₂)_(m)—R⁷;

or R⁸ and R⁹ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure;

R¹⁰ represents hydrogen, —CH₃, or —(CH₂)_(n)—CH₃;

Y¹ and Y² are each independently selected from OH and a group capable ofbeing hydrolyzed to OH, including cyclic derivatives where Y¹ and Y² areconnected via a ring having from 5 to 8 atoms in the ring structure;

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

In certain embodiments, the carbon bearing B(Y¹)(Y²) has the absolutestereochemical configuration of L-proline. In certain preferred suchembodiments, R² is selected from the group consisting of hydroxyl, loweralkyl, lower hydroxyalkyl and lower alkoxyalkyl. In more preferred suchembodiments, R² is located at the 5-position of the ring for lower alkyl(such as methyl), lower hydroxyalkyl (such as hydroxymethyl) and loweralkoxyalkyl or at the 4-position for hydroxyl. In most preferred suchembodiments, R² has a cis-stereochemical relationship to B(Y¹)(Y²).

Another aspect of the invention relates to compounds having a structureof Formula XXIV:

or a pharmaceutically acceptable salt thereof, wherein

A is a 3-8 membered heterocycle including the N and the Cα carbon;

W is a functional group which reacts with an active site residue of atargeted protease to form a covalent adduct;

R¹ is selected from the group consisting of hydrogen, a C-terminallylinked amino acid or peptide or analog thereof, and an amino protectinggroup; wherein optionally where applicable the bond between R¹ and the Nto which it is attached is a thioxamide bond;

R² represents one or more substitutions to the ring A, each of which isindependently a halogen, lower alkyl, lower alkenyl, lower alkynyl,carbonyl (such as a carboxyl, ester, formate, or ketone), thiocarbonyl(such as a thioester, thioacetate, or thioformate), amino, acylamino,amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R⁷,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R⁷, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

wherein at least one R² is selected from the group consisting of —OH,lower alkyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl,preferably at least one of lower alkyl (e.g., methyl), lower alkoxy,lower hydroxyalkyl (e.g., hydroxymethyl), and lower alkoxyalkyl;

R^(3a) is selected from the group consisting of hydrogen and asubstituent which does not conjugate the electron pair of the nitrogenfrom which it pends;

R^(4a) and R^(4b) are each independently selected from hydrogen, loweralkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,alkoxyl, carboxyl, carboxamide, carbonyl, and cyano, provided thateither both or neither of R^(4a) and R^(4b) are hydrogen;

R^(4c) is selected from the group consisting of halogen, amine, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, and cyano;

each R⁶ is independently selected from aryl, aralkyl, cycloalkyl,cycloalkenyl, and heterocyclyl;

z is zero or an integer in the range of 1 to 3;

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

In certain embodiments, W is selected from the group consisting of CNand B(Y¹)(Y²), wherein Y¹ and Y² are each independently or OH, or agroup capable of being hydrolyzed to OH, including cyclic derivativeswhere Y¹ and Y² are connected via a ring having from 5 to 8 atoms in thering structure. In certain embodiments, A is a five-membered ring, and Wis B(Y¹)(Y²).) In more certain embodiments, Cα has the absolutestereochemical configuration of L-proline.

In certain embodiments, A is a five-membered ring and R² is selectedfrom the group consisting of hydroxyl, lower alkyl, lower alkenyl, loweralkynyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl. Incertain preferred such embodiments, R² is selected from the groupconsisting of lower alkyl (such as methyl), lower hydroxyalkyl (such ashydroxymethyl) and lower alkoxyalkyl. In more preferred suchembodiments, R² is located at the 5-position of the ring.

In certain embodiments, A is a five-membered ring, and R² is selectedfrom the group consisting of hydroxyl, hydroxyl, lower alkyl, lowerhydroxyalkyl and lower alkoxyalkyl. In certain preferred suchembodiments, Cα has the absolute stereochemical configuration ofL-proline and R² is located at the 5-position of the ring for loweralkyl (such as methyl), lower hydroxyalkyl (such as hydroxymethyl) andlower alkoxyalkyl or at the 4-position for hydroxyl. In more preferredsuch embodiments, R² has a cis-stereochemical relationship to W.

Another aspect of the invention relates to compounds having a structureof Formula XXV:

or a pharmaceutically acceptable salt thereof, wherein

R¹, R², R^(3a), R^(4a), R^(4b), R^(4c) and W are as defined above forFormula XXIV, and p is an integer from 1 to 3. In certain embodiments, pis 1 and R^(3a) is hydrogen.

In certain embodiments, W is selected from the group consisting of CNand B(Y¹)(Y²), wherein Y¹ and Y² are each independently or OH, or agroup capable of being hydrolyzed to OH, including cyclic derivativeswhere Y¹ and Y² are connected via a ring having from 5 to 8 atoms in thering structure. In certain embodiments, W is B(Y¹)(Y²). In more certainembodiments, the carbon bearing W has the absolute stereochemicalconfiguration of L-proline.

In certain embodiments, R² is selected from the group consisting ofhydroxyl, lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lowerhydroxyalkyl, and lower alkoxyalkyl. In certain embodiments, R² isselected from the group consisting of lower hydroxyalkyl (such ashydroxymethyl) and lower alkoxyalkyl. In more preferred suchembodiments, p is 1 and R² is located at the 5-position of the ring.

In certain embodiments, R² is selected from the group consisting ofhydroxyl, lower alkyl, lower hydroxyalkyl and lower alkoxyalkyl. Incertain preferred such embodiments, p is 1, the carbon bearing W has theabsolute stereochemical configuration of L-proline and R² is located atthe 5-position of the ring for lower alkyl (such as methyl), lowerhydroxyalkyl (such as hydroxymethyl) and lower alkoxyalkyl or at the4-position for hydroxyl. In more preferred such embodiments, R² has acis-stereochemical relationship to W.

In certain embodiments, R² is azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

In certain embodiments, p is 1, the carbon bearing W has the absolutestereochemical configuration of L-proline and R² is located at the5-position of the ring.

Yet another aspect of the present invention relates to a compound havinga structure of Formula XXVI:

or a pharmaceutically acceptable salt thereof, wherein

A is a 3 to 8-membered heterocycle including the N and the Cα carbon;

B is a C₃₋₈ ring, or C₇₋₁₄ fused bicyclic or tricyclic ring system;

W is a functional group which reacts with an active site residue of atargeted protease to form a covalent adduct, as for example, —CN,—CH═NR⁵,

R¹ is selected from the group consisting of hydrogen, a C-terminallylinked amino acid or peptide or analog thereof, and an amino protectinggroup wherein optionally where applicable the bond between R¹ and the Nto which it is attached is a thioxamide bond;

R² represents one or more substitutions to the ring A, each of which isindependently a halogen, lower alkyl, lower alkenyl, lower alkynyl,carbonyl (such as a carboxyl, ester, formate, or ketone), thiocarbonyl(such as a thioester, thioacetate, or thioformate), amino, acylamino,amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R⁷,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R⁷, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

wherein at least one R² is selected from the group consisting of —OH,lower alkyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl,preferably at least one of lower alkyl (e.g., methyl), lower alkoxy,lower hydroxyalkyl (e.g., hydroxymethyl), and lower alkoxyalkyl;

R⁵ is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, —C(X¹)(X²)X³, —(CH₂)_(m)—R⁶, —(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl,—(CH₂)_(n)—O-alkenyl, —(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R⁶,—(CH₂)_(n)—SH, —(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl,—(CH₂)_(n)—S-alkynyl, —(CH₂)_(n)—S—(CH₂)_(m)—R⁶, —C(O)C(O)NH₂, and—C(O)C(O)OR⁷;

each R⁶ is independently selected from aryl, aralkyl, cycloalkyl,cycloalkenyl, and heterocyclyl;

each R⁷ is independently selected from hydrogen, alkyl, alkenyl, aryl,aralkyl, cycloalkyl, cycloalkenyl, and heterocycle;

R₈ represents hydrogen, —CH₃, or —(CH₂)_(n)—CH₃;

Y¹ and Y² are each independently selected from —OH, or a group capableof being hydrolyzed to a hydroxyl group, including cyclic derivativeswhere Y¹ and Y² are connected via a ring having from 5 to 8 atoms in thering structure (such as pinacol or the like),

R⁵⁰ is O or S;

R⁵¹ is selected from the group consisting of N₃, SH₂, NH₂, NO₂ or —OR⁷;

R⁵² represents hydrogen, a lower alkyl, an amine, —OR⁷, or apharmaceutically acceptable salt thereof, or R⁵¹ and R⁵² taken togetherwith the phosphorous atom to which they are attached complete aheterocyclic ring having from 5 to 8 atoms in the ring structure;

X¹ represents a halogen;

X² and X³ are each independently selected from hydrogen and halogen;

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

In certain embodiments, W is selected from the group consisting of CNand B(Y¹)(Y²), wherein Y¹ and Y² are each independently or OH, or agroup capable of being hydrolyzed to OH, including cyclic derivativeswhere Y¹ and Y² are connected via a ring having from 5 to 8 atoms in thering structure. In certain embodiments, A is a five-membered ring, and Wis B(Y¹)(Y²). In more certain embodiments, Cα has the absolutestereochemical configuration of L-proline.

In certain embodiments, A is a five-membered ring and R² is selectedfrom the group consisting of hydroxyl, lower alkyl, lower alkenyl, loweralkynyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl. Incertain preferred such embodiments, R² is selected from the groupconsisting of lower hydroxyalkyl (hydroxymethyl) and lower alkoxyalkyl.In more preferred such embodiments, R² is located at the 5-position ofthe ring.

In certain embodiments, A is a five-membered ring, and R² is selectedfrom the group consisting of hydroxyl, lower alkyl, lower hydroxyalkyland lower alkoxyalkyl. In certain preferred such embodiments, Cα has theabsolute stereochemical configuration of L-proline and R² is located atthe 5-position of the ring for lower alkyl (such as methyl), lowerhydroxyalkyl (such as hydroxymethyl) and lower alkoxyalkyl or at the4-position for hydroxyl. In more preferred such embodiments, R² has acis-stereochemical relationship to W.

In certain embodiments, R² is azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

In certain embodiments, p Cα has the absolute stereochemicalconfiguration of L-proline and R² is located at the 5-position of thering.

Another aspect of the invention relates to compounds having a structureof Formula XXVII:

or a pharmaceutically acceptable salt thereof, wherein

B, R¹, R² and W are as defined above for Formula XXVI, and p is aninteger from 1 to 3. In certain embodiments, p is 1.

In certain embodiments, W is selected from the group consisting of CNand B(Y¹)(Y²), wherein Y¹ and Y² are each independently or OH, or agroup capable of being hydrolyzed to OH, including cyclic derivativeswhere Y¹ and Y² are connected via a ring having from 5 to 8 atoms in thering structure. In certain embodiments, W is B(Y¹)(Y²). In more certainembodiments, the carbon bearing W has the absolute stereochemicalconfiguration of L-proline.

In certain embodiments, R² is selected from the group consisting ofhydroxyl, lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lowerhydroxyalkyl, and lower alkoxyalkyl. In certain preferred suchembodiments, R² is selected from the group consisting of lowerhydroxyalkyl (such as hydroxymethyl) and lower alkoxyalkyl. In morepreferred such embodiments, R² is located at the 5-position of the ring.

In certain embodiments, R² is selected from the group consisting ofhydroxyl, lower alkyl, lower hydroxyalkyl and lower alkoxyalkyl. Incertain preferred such embodiments, p is 1, the carbon bearing W has theabsolute stereochemical configuration of L-proline and R² is located atthe 4-position of the ring for hydroxyl or at the 5-position for loweralkyl (such as methyl), lower hydroxyalkyl (such as hydroxymethyl) andlower alkoxyalkyl. In more preferred such embodiments, R² has acis-stereochemical relationship to W.

In certain embodiments, R² is azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

In certain embodiments, p is 1, the carbon bearing W has the absolutestereochemical configuration of L-proline and R² is located at the5-position of the ring.

Another aspect of the invention relates to compounds having a structureof Formula XXVIII:

or a pharmaceutically acceptable salt thereof, wherein

A is a 4-8 membered heterocycle including the N and the Cα carbon;

W is a functional group which reacts with an active site residue of thetargeted protease to form a covalent adduct, as for example, —CN,—CH═NR⁵,

R¹ represents a C-terminally linked peptide or peptide analog which is asubstrate for an activating enzyme; wherein optionally the bond betweenR¹ and the N to which it is bonded is a thioxamide bond;

R² represents one or more substitutions to the ring A, each of which isindependently a halogen, lower alkyl, lower alkenyl, lower alkynyl,carbonyl (such as a carboxyl, ester, formate, or ketone), thiocarbonyl(such as a thioester, thioacetate, or thioformate), amino, acylamino,amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R⁷,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R⁷, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

wherein at least one R² is selected from the group consisting of —OH,lower alkyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl,preferably at least one of lower alkyl (e.g., methyl), lower alkoxy,lower hydroxyalkyl (e.g., hydroxymethyl), and lower alkoxyalkyl;

R³ is selected from the group consisting of hydrogen and a substituentwhich does not conjugate the electron pair of the nitrogen to which itis attached, such as a lower alkyl;

R⁴ is selected from the group consisting of hydrogen and a smallhydrophobic group such as a halogen, lower alkyl, lower alkenyl, orlower alkynyl;

R⁵ is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, —C(X¹)(X²)X³, —(CH²)^(m)—R⁶, —(CH²)^(n)—OH, —(CH²)^(n)—O-alkyl,—(CH²)^(n)—O-alkenyl, —(CH²)^(n)—O-alkynyl, —(CH²)^(n)—O—(CH²)^(m)—R⁶,—(CH²)^(n)—SH, —(CH²)^(n)—S-alkyl, —(CH²)^(n)—S-alkenyl,—(CH²)^(n)—S-alkynyl, —(CH²)^(n)—S—(CH²)^(m)—R⁶, —C(O)C(O)NH²,—C(O)C(O)OR⁷;

R⁶ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R⁷ represents, for each occurrence, hydrogen, or a substituted orunsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl,or heterocycle;

R₈ represents hydrogen, —CH₃, or —(CH₂)_(n)—CH₃; and

Y¹ and Y² are independently or together OH or a group capable of beinghydrolyzed to a hydroxyl group, including cyclic derivatives where Y¹and Y² are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R⁵⁰ is O or S;

R⁵¹ is selected from the group consisting of N₃, SH₂, NH₂, NO₂ and —OR⁷;

R⁵² is selected from the group consisting of hydrogen, lower alkyl,amine, —OR⁷, or a pharmaceutically acceptable salt thereof; or

R⁵¹ and R⁵² taken together with the phosphorous atom to which they areattached complete a heterocyclic ring having from 5 to 8 atoms in thering structure;

X¹ is a halogen;

X² and X³ are each independently selected from hydrogen and halogen;

m is zero or an integer in the range of 1 to 8; and

n is an integer in the range of 1 to 8.

In certain embodiments, W is selected from the group consisting of CNand B(Y¹)(Y²). In certain embodiments, A is a five-membered ring, and Wis B(Y¹)(Y²).) In more certain embodiments, Cα has the absolutestereochemical configuration of L-proline.

In certain embodiments, A is a five-membered ring, Z is C, and R² isselected from the group consisting of hydroxyl, lower alkyl, loweralkenyl, lower alkynyl, lower alkoxy, lower hydroxyalkyl, and loweralkoxyalkyl. In certain preferred such embodiments, R² is selected fromthe group consisting of lower hydroxyalkyl (such as hydroxymethyl) andlower alkoxyalkyl. In more preferred such embodiments, R² is located atthe 5-position of the ring.

In certain embodiments, A is a five-membered ring and R² is selectedfrom the group consisting of hydroxyl, lower alkyl, lower hydroxyalkyland lower alkoxyalkyl. In certain preferred such embodiments, Cα has theabsolute stereochemical configuration of L-proline and R² is located atthe 4-position of the ring for hydroxyl or at the 5-position for loweralkyl, lower hydroxyalkyl and lower alkoxyalkyl. In more preferred suchembodiments, R² has a cis-stereochemical relationship to W.

In certain embodiments, R² is azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

In certain embodiments, Cα has the absolute stereochemical configurationof L-proline. In more certain embodiments, R² is located at the5-position of the ring.

One aspect of the invention relates to compounds having a structure ofFormula XXIX:

or a pharmaceutically acceptable salt thereof, wherein

L is absent or is —XC(O)—;

R¹ is selected from the group consisting of H, lower alkyl, lower acyl,lower aralkyl, lower aracyl, lower heteroaracyl, carbocyclyl, aryl, andArSO₂—; wherein optionally when L is absent the bond between R¹ and theN to which it is bonded is a thioxamide bond;

R² represents one or more substitutions to the ring A, each of which isindependently a halogen, lower alkyl, lower alkenyl, lower alkynyl,carbonyl (such as a carboxyl, ester, formate, or ketone), thiocarbonyl(such as a thioester, thioacetate, or thioformate), amino, acylamino,amido, nitro, sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R⁷,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R⁷, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, or —(CH₂)_(n)—S—(CH₂)_(m)—R⁷, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

wherein at least one R² is selected from the group consisting of —OH,lower alkyl, lower alkoxy, lower hydroxyalkyl, and lower alkoxyalkyl,preferably at least one of lower alkyl (e.g., methyl), lower alkoxy,lower hydroxyalkyl (e.g., hydroxymethyl), and lower alkoxyalkyl;

R³ is selected from the group consisting of hydrogen, lower alkyl, lowerhydroxyalkyl, lower thioalkyl, and lower aralkyl;

R⁴ is selected from the group consisting of H and lower alkyl, or R¹ andR⁴ together are phthaloyl, thereby forming a ring;

R⁶ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R₈ represents hydrogen, —CH₃, or —(CH₂)_(n)—CH₃;

W is selected from the group consisting of B(Y¹)(Y²) and CN;

Y¹ and Y² are independently selected from OH or a group that ishydrolysable to an OH, or together with the boron atom to which they areattached form a 5- to 8-membered ring that is hydrolysable to OH;

X is selected from the group consisting of O and NH.

In certain embodiments, W is B(Y¹)(Y²). In certain embodiments, R² isselected from the group consisting of hydroxyl, lower alkyl, loweralkenyl, lower alkynyl, lower alkoxy, lower hydroxyalkyl, and loweralkoxyalkyl. In more preferred such embodiments, R² is selected from thegroup consisting of lower hydroxyalkyl and lower alkoxyalkyl. In morepreferred such embodiments, R² is located at the 5-position of the ring.

In certain embodiments, R² is selected from the group consisting ofhydroxyl, lower alkyl, lower hydroxyalkyl and lower alkoxyalkyl. Incertain preferred such embodiments, Cα has the absolute stereochemicalconfiguration of L-proline and R² is located at the 4-position of thering for hydroxyl or at the 5-position for lower alkyl, lowerhydroxyalkyl and lower alkoxyalkyl. In more preferred such embodiments,R² has a cis-stereochemical relationship to W.

Embodiment F

A representative class of compounds for use in the method of the presentinvention are represented by formula XXX:

wherein

R¹ is selected from the group consisting of H, alkyl, alkoxy, alkenyl,alkynyl, amino, alkylamino, acylamino, cyano, sulfonylamino, acyloxy,aryl, cycloalkyl, heterocyclyl, heteroaryl, and polypeptide chains of 1to 8 amino acid residues;

R² is selected from the group consisting of H, lower alkyl, and aralkyl;

R³ is selected from the group consisting of

(a) lower alkyl;

(b) R^(a)R^(b)N(CH₂)_(m)— wherein

R^(a) is a pyridinyl or pyrimidinyl moiety optionally substituted with(C₁₋₄)alkyl, (C₁₋₄)alkoxy, halogen, trifluoromethyl, cyano or nitro; orphenyl optionally mono- or independently disubstituted with (C₁₋₄)alkyl,(C₁₋₄)alkoxy or halogen;

R^(b) is hydrogen or (C₁₋₈)alkyl and m is 2 or 3;

(c) (C₃₋₁₂)cycloalkyl optionally monosubstituted in the 1-position with(C₁₋₃)hydroxyalkyl;

(d) R^(c)(CH₂)_(n)— wherein either R^(c) is phenyl optionallysubstituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy, halogen or phenylthiooptionally monosubstituted in the phenyl ring with hydroxymethyl; or is(C₁₋₈)alkyl; a [3.1.1]bicyclic carbocyclic moiety optionally substitutedwith (C₁₋₈)alkyl; a pyridinyl or naphthyl moiety optionally substitutedwith (C₁₋₄)alkyl, (C₁₋₄)alkoxy or halogen; cyclohexene; or adamantyl andn is 1 to 3; or R^(c) is phenoxy optionally substituted with(C₁₋₄)alkyl, (C₁₋₄)alkoxy or halogen and n is 2 or 3;

(e) (R^(d))₂CH(CH₂)₂— wherein each R^(d) independently is phenyloptionally substituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy, or halogen;

(f) R^(e)(CH₂)_(p)— wherein R^(e) is 2-oxopyrrolidinyl or (C₂₋₄)alkoxyand p is 2 to 4; and

(g) R^(g) wherein R^(g) is: indanyl; a pyrrolidinyl or piperidinylmoiety optionally substituted with benzyl; a [2.2.1]- or [3.1.1]bicycliccarbocyclic moiety optionally substituted with (C₁₋₈)alkyl; adamantyl;or (C₁₋₈)alkyl optionally substituted with hydroxy, hydroxymethyl orphenyl optionally substituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy or halogen;

R⁴ is selected from the group consisting of H, halogen, and lower alkyl;

R⁵ is selected from the group consisting of H, halogen lower alkyl, andaralkyl, preferably H or lower alkyl;

R⁶ is a functional group that reacts with an active site residue of thetargeted protease to form a covalent adduct;

R⁷ is selected from the group consisting of alkyl, alkoxy, alkenyl,alkynyl, aminoalkyl, aminoacyl, acyloxy, aryl, aralkyl, cycloalkyl,heterocyclyl, heteroaryl, or heteroaralkyl;

R⁸ is selected from the group consisting of H, aryl, alkyl, aralkyl,cycloalkyl, heterocyclyl, heteroaryl, heteroaralkyl, and polypeptidechains of 1 to 8 amino acid residues;

L is absent or is selected from the group consisting of alkyl, alkenyl,alkynyl, (CH₂)_(m)—O—(CH₂)_(m),

—(CH₂)_(m)NR₂(CH₂)_(m)—, and —(CH₂)_(m)S(CH₂)_(m)—;

X is absent or is selected from the group consisting of —N(R⁸)—, —O—,and —S—;

Y is absent or is selected from the group consisting of —C(═O)—,—C(═S)—, and —SO₂—;

m is, independently for each occurrence, an integer from 0 to 10; and

n is an integer from 1 to 6.

In certain embodiments, R¹ is H or lower alkyl and R⁴ and R⁵ are bothhydrogen. In certain such embodiments, R³ is lower alkyl. In certainpreferred such embodiments, R³ is selected from the group consisting ofmethyl, ethyl, and isopropyl.

In certain embodiments R³ is a substituted lower alkyl. In certain suchembodiments, R³ is substituted with a group selected from halogen,hydroxyl, carbonyl, thiocarbonyl, alkoxy, amino, amido, amidine, cyano,nitro, alkylthio, heterocyclyl, aryl, and heteroaryl.

In certain embodiments, X, Y, and L are absent and R¹ is a polypeptidechain of 2 to 8 amino acid residues. In certain such embodiments, R¹ isa polypeptide chain of 2 amino acid residues. In such embodiments, thebond between R¹ and N may be a thioxamide bond.

In certain other embodiments, R⁶ is selected from the group consistingof boronic acid, CN, —SO₂Z¹, —P(═O)Z¹, —P(R⁹)R¹⁰R¹¹, —C(═NH)NH₂,—CH═NR¹², or —C(═O)—R¹² wherein

R⁹ is selected from the group consisting of O and S;

R¹⁹ is selected from the group consisting of N₃, SH₂, NH₂, NO₂, andOLR¹³, and

R¹¹ is selected from the group consisting of lower alkyl, amino, andOLR¹³, or a pharmaceutically acceptable salt thereof; or

R¹⁰ and R¹¹, together with the phosphorus to which they are attached,form a 5- to 8-membered heterocyclic ring;

R¹² is selected from the group consisting of H, alkyl, alkenyl, alkynyl,NH₂, —(CH₂)_(p)—R¹³, —(CH₂)_(q)—OH, —(CH₂)_(q)—O-alkyl,—(CH₂)_(q)—O-alkenyl, —(CH₂)_(q)—O-alkynyl, —(CH₂)_(q)—O—(CH₂)_(p)—R¹³,—(CH₂)_(q)—SH, —(CH₂)_(q)—S-alkyl, —(CH₂)_(q)—S-alkenyl,—(CH₂)_(q)—S-alkynyl, —(CH₂)_(q)—S—(CH₂)_(p)—R¹³, —C(O)NH₂, —C(O)OR¹⁴,and C(Z¹)(Z²)(Z³);

R¹³ is selected from the group consisting of H, alkyl, alkenyl, aryl,heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclyl;

R¹⁴ is selected from the group consisting of H, alkyl, alkenyl, andLR¹³;

Z¹ is a halogen;

Z² and Z³ are independently selected from H and halogen;

p is, independently for each occurrence, an integer from 0 to 8; and

q is, independently for each occurrence, an integer from 1 to 8.

In certain embodiments, R⁶ is selected from the group consisting of CN,CHO, and C(═O)C(Z¹)(Z²)(Z³), wherein Z¹ is a halogen and Z² and Z³ areindependently selected from H and halogen. In another embodiment, R⁶ isselected from the group consisting of C(═O)C(Z¹)(Z²)(Z³), wherein Z¹ isfluorine and Z² and Z³ are independently selected from H and fluorine.

In certain embodiments, R⁶ is a group —B(Y¹)(Y²), wherein Y¹ and Y² areindependently OH or a group that is hydrolysable to a boronic acid, ortogether with the boron atom to which they are attached form a 5- to8-membered ring that is hydrolysable to a boronic acid.

In certain embodiments, exemplary compounds of the present inventioninclude:

or enantiomers or diastereomers thereof.

Also included are compounds of Formulas I-XXX, wherein one or more amidegroups are replaced by one or more thioxamide groups.

Also included are such peptidomimetics as olefins, phosphonates,aza-amino acid analogs and the like.

Also deemed as equivalents are any compounds which can be hydrolyticallyconverted into any of the aforementioned compounds including boronicacid esters and halides, and carbonyl equivalents including acetals,hemiacetals, ketals, and hemiketals, and cyclic dipeptide analogs.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the subject compound which contain a basic or acidmoiety by conventional chemical methods. Generally, the salts areprepared by reacting the free base or acid with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidor base in a suitable solvent. The pharmaceutically acceptable salts ofthe acids of the subject compounds are also readily prepared byconventional procedures such as treating an acid of the presentcompounds with an appropriate amount of a base such as an alkali oralkaline earth methyl hydroxide (e.g., sodium, potassium, lithium,calcium or magnesium) or an organic base such as an amine, piperidine,pyrrolidine, benzylamine and the like, or a quaternary ammoniumhydroxide such as tetramethylammonium hydroxide and the like.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit proteolysis ofGLP-1 or other peptide hormone or precursor thereof), wherein one ormore simple variations of substituents are made which do not adverselyaffect the efficacy of the compound in use in the contemplated method.In general, the compounds of the present invention may be prepared bythe methods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

In certain embodiments, the compounds are DPIV inhibitors with a K_(i)for DPIV inhibition of 10 nm or less, more preferably of 1.0 nm or less,and even more preferably of 0.1 or even 0.01 nM or less. Indeed,inhibitors with K_(i) values in the picomolar and even femtomolar rangeare contemplated.

Another aspect of the present invention relates to pharmaceuticalcompositions of the dipeptidylpeptidase inhibitors disclosed herein,particularly compound(s) and their uses in treating and/or preventing(inhibiting) disorders which can be improved by altering the homeostasisof peptide hormones. In a certain embodiment, the compounds havehypoglycemic and antidiabetic activities, and can be used in thetreatment of disorders marked by aberrant glucose metabolism (includingstorage). In particular embodiments, the compositions of the subjectmethods are useful as insulinotropic agents, or to potentiate theinsulinotropic effects of such molecules as GLP-1. In this regard, thepresent method can be useful for the treatment and/or prophylaxis of avariety of disorders, including one or more of: hyperlipemia,hyperglycemia, obesity, glucose tolerance insufficiency, insulinresistance, and diabetic complications.

For instance, in certain embodiments the method involves administrationof a compound(s), preferably at a predetermined interval(s) during a24-hour period, in an amount effective to improve one or more aberrantindices associated with glucose metabolism disorders (e.g., glucoseintolerance, insulin resistance, hyperglycemia, hyperinsulinemia, andType II diabetes). The effective amount of the compound may be about0.01, 0.1, 1, 10, 30, 50, 70, 100, 150, 200, 500, or 1000 mg/kg of thesubject.

(ii). Agonism of GLP-1 Effects

The compounds useful in the subject methods possess, in certainembodiments, the ability to lower blood glucose levels, to relieveobesity, to alleviate impaired glucose tolerance, to inhibit hepaticglucose neogenesis, and to lower blood lipid levels and to inhibitaldose reductase. They are thus useful for the prevention and/or therapyof hyperglycemia, obesity, hyperlipidemia, diabetic complications(including retinopathy, nephropathy, neuropathy, cataracts, coronaryartery disease and arteriosclerosis), and furthermore forobesity-related hypertension and osteoporosis.

Diabetes mellitus is a disease characterized by hyperglycemia occurringfrom a relative or absolute decrease in insulin secretion, decreasedinsulin sensitivity, or insulin resistance. The morbidity and mortalityof this disease result from vascular, renal, and neurologicalcomplications. An oral glucose tolerance test is a clinical test used todiagnose diabetes. In an oral glucose tolerance test, a patient'sphysiological response to a glucose load or challenge is evaluated.After ingesting the glucose, the patient's physiological response to theglucose challenge is evaluated. Generally, this is accomplished bydetermining the patient's blood glucose levels (the concentration ofglucose in the patient's plasma, serum, or whole blood) for severalpredetermined points in time.

In one embodiment, the present invention provides a method for agonizingthe action of GLP-1. It has been determined that isoforms of GLP-1(GLP-1(7-37) and GLP-1(7-36)), which are derived from preproglucagon inthe intestine and the hind brain, have insulinotropic activity, i.e.,they modulate glucose metabolism. DPIV cleaves the isoforms to inactivepeptides. Thus, in certain embodiments, compound(s) of the presentinvention can agonize insulinotropic activity by interfering with thedegradation of bioactive GLP-1 peptides.

(iii). Agonism of the Effects of Other Peptide Hormones

In another embodiment, the subject agents can be used to agonize (e.g.,mimic or potentiate) the activity of peptide hormones, e.g., GLP-2, GIPand NPY.

To illustrate further, the present invention provides a method foragonizing the action of GLP-2. It has been determined that GLP-2 acts asa trophic agent, to promote growth of gastrointestinal tissue. Theeffect of GLP-2 is marked particularly by increased growth of the smallbowel, and is therefore herein referred to as an “intestinotrophic”effect. DPIV is known to cleave GLP-2 into a biologically inactivepeptide. Thus, in one embodiment, inhibition of DPIV interferes with thedegradation of GLP-2, and thereby increases the plasma half-life of thathormone.

In still other embodiments, the subject method can be used to increasethe half-life of other proglucagon-derived peptides, such as glicentin,oxyntomodulin, glicentin-related pancreatic polypeptide (GRPP), and/orintervening peptide-2 (IP-2). For example, glicentin has beendemonstrated to cause proliferation of intestinal mucosa and alsoinhibits a peristalsis of the stomach, and has thus been elucidated asuseful as a therapeutic agent for digestive tract diseases, thus leadingto the present invention.

Thus, in one aspect, the present invention relates to therapeutic andrelated uses of compound(s) for promoting the growth and proliferationof gastrointestinal tissue, most particularly small bowel tissue. Forinstance, the subject method can be used as part of a regimen fortreating injury, inflammation, or resection of intestinal tissue, e.g.,where enhanced growth and repair of the intestinal mucosal epithelial isdesired.

With respect to small bowel tissue, such growth is measured convenientlyas an increase in small bowel mass and length, relative to an untreatedcontrol. The effect of compounds on small bowel also manifests as anincrease in the height of the crypt plus villus axis. Such activity isreferred to herein as an “intestinotrophic” activity. The efficacy ofthe subject method may also be detectable as an increase in crypt cellproliferation and/or a decrease in small bowel epithelium apoptosis.These cellular effects may be noted most significantly in relation tothe jejunum, including the distal jejunum and particularly the proximaljejunum, and also in the distal ileum. A compound is considered to have“intestinotrophic effect” if a test animal exhibits significantlyincreased small bowel weight, increased height of the crypt plus villusaxis or increased crypt cell proliferation, or decreased small bowelepithelium apoptosis when treated with the compound (or geneticallyengineered to express it themselves). A model suitable for determiningsuch gastrointestinal growth is described by U.S. Pat. No. 5,834,428.

In general, patients who would benefit from either increased smallintestinal mass and consequent increased small bowel mucosal functionare candidates for treatment by the subject method. Particularconditions that may be treated include the various forms of sprue,including celiac sprue which results from a toxic reaction to α-gliadinfrom wheat, and is marked by a tremendous loss of villae of the bowel;tropical sprue which results from infection and is marked by partialflattening of the villae; hypogammaglobulinemic sprue which is observedcommonly in patients with common variable immunodeficiency orhypogammaglobulinemia and is marked by significant decrease in villusheight. The therapeutic efficacy of the treatment may be monitored byenteric biopsy to examine the villus morphology, by biochemicalassessment of nutrient absorption, by patient weight gain, or byamelioration of the symptoms associated with these conditions. Otherconditions that may be treated by the subject method, or for which thesubject method may be useful prophylactically, include radiationenteritis, infectious or post-infectious enteritis, regional enteritis(Crohn's disease), small intestinal damage due to toxic or otherchemotherapeutic agents, and patients with short bowel syndrome.

More generally, the present invention provides a therapeutic method fortreating digestive tract diseases. The term “digestive tract” as usedherein means a tube through which food passes, including stomach andintestine. The term “digestive tract diseases” as used herein meansdiseases accompanied by a qualitative or quantitative abnormality in thedigestive tract mucosa, which include, e.g., ulceric or inflammatorydisease; congenital or acquired digestion and absorption disorderincluding malabsorption syndrome; disease caused by loss of a mucosalbarrier function of the gut; and protein-losing gastroenteropathy. Theulceric disease includes, e.g., gastric ulcer, duodenal ulcer, smallintestinal ulcer, colonic ulcer, and rectal ulcer. The inflammatorydisease include, e.g., esophagitis, gastritis, duodenitis, enteritis,colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiationenteritis, radiation colitis, radiation proctitis, enteritis, andmedicamentosa. The malabsorption syndrome includes the essentialmalabsorption syndrome such as disaccharide-decomposing enzymedeficiency, glucose-galactose malabsorption, fructose malabsorption;secondary malabsorption syndrome, e.g., the disorder caused by a mucosalatrophy in the digestive tract through the intravenous or parenteralnutrition or elemental diet, the disease caused by the resection andshunt of the small intestine such as short gut syndrome, cul-de-sacsyndrome; and indigestible malabsorption syndrome, such as the diseasecaused by resection of the stomach, e.g., dumping syndrome.

The term “therapeutic agent for digestive tract diseases” as used hereinmeans the agents for the prevention and treatment of the digestive tractdiseases, which include, e.g., the therapeutic agent for digestive tractulcer, the therapeutic agent for inflammatory digestive tract disease,the therapeutic agent for mucosal atrophy in the digestive tract, thetherapeutic agent for a digestive tract wound, the amelioration agentfor the function of the digestive tract including the agent for recoveryof the mucosal barrier function, and the amelioration agent fordigestive and absorptive function. Ulcers include digestive ulcers anderosions, and acute ulcers, namely acute mucosal lesions.

The subject method, because of promoting proliferation of intestinalmucosa, can be used in the treatment and prevention of pathologicconditions of insufficiency in digestion and absorption, that is,treatment and prevention of mucosal atrophy, or treatment of hypoplasiaof the digestive tract tissues and decrease in these tissues by surgicalremoval as well as improvement of digestion and absorption. Further, thesubject method can be used in the treatment of pathologic mucosalconditions due to inflammatory diseases such as enteritis, Crohn'sdisease, and ulceric colitis and also in the treatment of reduction infunction of the digestive tract after operation, for example, in dampingsyndrome as well as in the treatment of duodenal ulcer in conjunctionwith the inhibition of peristalsis of the stomach and rapid migration offood from the stomach to the jejunum. Furthermore, glicentin caneffectively be used in promoting cure of surgical invasion as well as inimproving functions of the digestive tract. Thus, the present inventionalso provides a therapeutic agent for atrophy of the digestive tractmucosa, a therapeutic agent for wounds in the digestive tract and a drugfor improving functions of the digestive tract which comprise glicentinas active ingredients.

Likewise, the compound(s) of the subject invention can be used to alterthe plasma half-life of secretin, VIP, PHI, PACAP, GIP, and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

Neuropeptide Y (NPY) is believed to act in the regulation vascularsmooth muscle tone, as well as regulation of blood pressure. NPY alsodecreases cardiac contractility. NPY is also the most powerful appetitestimulant known (Wilding et al., J. Endocrinology 1992, 132, 299-302).The centrally evoked food intake (appetite stimulation) effect ispredominantly mediated by NPY Y1 receptors and causes increase in bodyfat stores and obesity (Stanley et al., Physiology and Behavior 1989,46, 173-177).

According to the present invention, a method for treatment of anorexiacomprises administering to a host subject an effective amount of acompound(s) to stimulate the appetite and increase body fat stores whichthereby substantially relieves the symptoms of anorexia.

A method for treatment of hypotension comprises administering to a hostsubject an effective amount of a compound(s) of the present invention tomediate vasoconstriction and increase blood pressure which therebysubstantially relieves the symptoms of hypotension.

DPIV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin (Kubiak et al. Peptide Res. 1994, 7, 153). GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

(iv). Assays of Insulinotropic Activity

In selecting a compound suitable for use in the subject method, it isnoted that the insulinotropic property of a compound may be determinedby providing that compound to animal cells, or injecting that compoundinto animals and monitoring the release of immunoreactive insulin (IRI)into the media or circulatory system of the animal, respectively. Thepresence of IRI can be detected through the use of a radioimmunoassaywhich can specifically detect insulin.

The db/db mouse is a genetically obese and diabetic strain of mouse. Thedb/db mouse develops hyperglycemia and hyperinsulinemia concomitant withits development of obesity and thus serves as a model of obese type 2diabetes (NIDDM). The db/db mice can be purchased from, for example, TheJackson Laboratories (Bar Harbor, Me.). In an exemplary embodiment, fortreatment of the mice with a regimen including a compound(s) or control,sub-orbital sinus blood samples are taken before and at some time (e.g.,60 min) after dosing of each animal. Blood glucose measurements can bemade by any of several conventional techniques, such as using a glucosemeter. The blood glucose levels of the control and compound(s) dosedanimals are compared

The metabolic fate of exogenous GLP-1 can also be followed in bothnondiabetic or type II diabetic subjects, and the effect of a candidatecompound(s) determined. For instance, a combination of high-pressureliquid chromatography (HPLC), specific radioimmunoassays (RIAs), and anenzyme-linked immunosorbent assay (ELISA), can be used, whereby intactbiologically active GLP-1 and its metabolites can be detected. See, forexample, Deacon et al. Diabetes, 1995, 44, 1126-1131. To illustrate,after GLP-1 administration, the intact peptide can be measured using anNH₂-terminally directed RIA or ELISA, while the difference inconcentration between these assays and a COOH-terminal-specific RIAallowed determination of NH₂-terminally truncated metabolites. Withoutcompound, subcutaneous GLP-1 is rapidly degraded in a time-dependentmanner, forming a metabolite which co-elutes on HPLC with GLP-1(9-36)amide and has the same immunoreactive profile. For instance, 30 minafter subcutaneous GLP-1 administration to diabetic patients (n=8), themetabolite accounted for 88.5+1.9% of the increase in plasmaimmunoreactivity determined by the COOH-terminal RIA, which was higherthan the levels measured in healthy subjects (78.4+3.2%; n=8; P<0.05).See Deacon et al., supra. Intravenously infused GLP-1 was alsoextensively degraded.

(v). Conjoint Administration

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the compound.Such conjoint treatment may be achieved by way of the simultaneous,sequential, or separate dosing of the individual components of thetreatment.

In one embodiment, a compound(s) is conjointly administered with insulinor other insulinotropic agents, such as GLP-1, peptide hormones, such asGLP-2, GIP, or NPY, or a gene therapy vector which causes the ectopicexpression of said agents and peptide hormones. In certain embodiments,said agents or peptide hormones may be variants of a naturally occurringor synthetic peptide hormone, wherein one or more amino acids have beenadded, deleted, or substituted.

In another illustrative embodiment, the compounds can be conjointlyadministered with an M1 receptor antagonist. Cholinergic agents arepotent modulators of insulin release that act via muscarinic receptors.Moreover, the use of such agents can have the added benefit ofdecreasing cholesterol levels, while increasing HDL levels. Suitablemuscarinic receptor antagonists include substances that directly orindirectly block activation of muscarinic cholinergic receptors.Preferably, such substances are selective (or are used in amounts thatpromote such selectivity) for the M1 receptor. Non-limiting examplesinclude quaternary amines (such as methantheline, ipratropium, andpropantheline), tertiary amines (e.g., dicyclomine and scopolamine), andtricyclic amines (e.g., telenzepine). Pirenzepine and methyl scopolamineare preferred. Other suitable muscarinic receptor antagonists includebenztropine (commercially available as COGENTIN from Merck),hexahydro-sila-difenidol hydrochloride (HHSID hydrochloride disclosed inLambrecht et al. Trends in Pharmacol. Sci. 1989, 10(Suppl), 60;(+/−)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate(QNX-hemioxalate; Birdsall et al., Trends in Pharmacol. Sci. 1983, 4,459; telenzepine dihydrochloride (Coruzzi et al. Arch. Int. Pharmacodyn.Ther. 1989, 302, 232; and Kawashima et al. Gen. Pharmacol. 1990, 21,17), and atropine. The dosages of such muscarinic receptor antagonistswill be generally subject to optimization as outlined above. In the caseof lipid metabolism disorders, dosage optimization may be necessaryindependent of whether administration is timed by reference to the lipidmetabolism responsiveness window or not.

In terms of regulating insulin and lipid metabolism and reducing theforegoing disorders, the compound(s) may also act synergistically withprolactin inhibitors such as d2 dopamine agonists (e.g., bromocriptine).Accordingly, the subject method can include the conjoint administrationof such prolactin inhibitors as prolactin-inhibiting ergo alkaloids andprolactin-inhibiting dopamine agonists. Examples of suitable compoundsinclude 2-bromo-alpha-ergocriptine,6-methyl-8-beta-carbobenzyloxyaminoethyl-10-alpha-ergoline,8-acylaminoergolines, 6-methyl-8-alpha-(N-acyl)amino-9-ergoline,6-methyl-8-alpha-(N-phenylacetyl)amino-9-ergoline, ergocornine,9,10-dihydroergocornine, D-2-halo-6-alkyl-8-substituted ergolines,D-2-bromo-6-methyl-8-cyanomethylergoline, carbidopa, benserazide, andother dopadecarboxylase inhibitors, L-dopa, dopamine, and non toxicsalts thereof.

The compound(s) used according to the invention can also be usedconjointly with agents acting on the ATP-dependent potassium channel ofthe β-cells, such as glibenclamide, glipizide, gliclazide, and AG-EE 623ZW. The compound(s) may also advantageously be applied in combinationwith other oral agents such as metformin and related compounds orglucosidase inhibitors as, for example, acarbose.

(vi). Hematopoietic Agonists.

In still another aspect, the present invention provides a method forstimulating hematopoietic cells in culture or in vivo. In certainembodiments, the subject DPP IV pro-inhibitors include an address moietythat is a substrate for a protease that is expressed in bone marrow.

According to one aspect of the invention, a method for stimulatinghematopoietic cells in vitro is provided. The method involves (1)contacting the hematopoietic cells with a sufficient amount of an DPP IVpro-inhibitor to increase the number of hematopoietic cells and/or thedifferentiation of such hematopoietic cells relative to the number anddifferentiation of hematopoietic cells.

One important aspect of the invention involves restoring or preventing adeficiency in hematopoietic cell number in a subject. Such deficienciescan arise, for example, from genetic abnormalities, from disease, fromstress, from chemotherapy (e.g. cytotoxic drug treatment, steroid drugtreatment, immunosuppressive drug treatment, etc.) and from radiationtreatment.

The pro-inhibitors of the invention can be administered alone, or incombination with additional agents for treating the condition, e.g., adifferent agent which stimulates activation or proliferation of saidlymphocytes or hematopoietic cells. For example, the pro-inhibitors canbe administered in conjunction with exogenous growth factors andcytokines which are specifically selected to achieve a particularoutcome. For example, if it is desired to stimulate a particularhematopoietic cell type, then growth factors and cytokines whichstimulate proliferation and differentiation of such cell type are used.

Thus, it is known that interleukins-1,2,3,4,5,6,7,9,10, 11, 12, 13, and17 are involved in lymphocyte differentiation. Interleukins 3 and 4 areinvolved in mast cell differentiation. Granulocyte macrophage colonystimulating factor (GMCSF), interleukin-3 and interleukin-5 are involvedin the eosinophil differentiation. GMCSF, macrophage colony stimulatingfactor (MCSF) and IL-3 are involved in macrophage differentiation.

GMCSF, GCSF and IL-3 are involved in neutrophil differentiation. GMSCF,IL-3, IL-6, IL-11 and TPO are involved in platelet differentiation. Flt3Ligand is involved in dendritic cell growth. GMCSF, IL-3, anderythropoietin are involved in erythrocyte differentiation.

Finally, the self-renewal of primitive, pluripotent progenitor cellscapable of sustaining hematopoiesis requires SCF, Flt3 Ligand, G-CSF,IL-3, IL-6 and IL-11. Various combinations for achieving a desiredresult will be apparent to those of ordinary skill in the art.

(vii). Proteasome Inhibitors.

In other embodiments, the pro-soft inhibitors produce inhibitor moietiesthat are potent and highly selective proteasome inhibitors and can beemployed to inhibit proteasome function Inhibition of proteasomefunction has a number of practical therapeutic and prophylacticapplications. However, because the proteasome is ubiquitous to livingcells, there is a desire to provide embodiments of the subjectpro-inhibitor that release a proteasome inhibitor using an addressmoiety that is cleaved at or in proximity to the intended target cells.For instance, the proteasome pro-inhibitors embodiments can includeaddress moieties that are substrates for proteases that are expressed intumors or other cells which are undergoing unwanted proliferation, orexpressed in the tissue surrounding the tumor or other targetproliferating cells. For instance, the address moiety can be a substratefor a protease expressed in the stromal layer adjacent a tumor.

In certain embodiments, the proteasome pro-inhibitors of the presentinvention provide a method of reducing the rate of degradation of p53and other tumor suppressors.

Such pro-inhibitors are contemplated as possessing important practicalapplication in treating cell proliferative diseases, such as cancer,restenosis and psoriasis.

In certain embodiments, proteasome pro-inhibitors can be used to inhibitthe processing of internalized cellular or viral antigens into antigenicpeptides that bind to MHC-I molecules in an animal, and are thereforeuseful for treating autoimmune diseases and preventing rejection offoreign tissues, such as transplanted organs or grafts.

Finally, the present invention relates to the use of proteasomepro-inhibitors for treating specific conditions in animals that aremediated or exacerbated, directly or indirectly, by proteasomefunctions. These conditions include inflammatory conditions, such astissue rejection, organ rejection, arthritis, infection, dermatoses,inflammatory bowel disease, asthma, osteoporosis, osteoarthritis andautoimmune disease such as lupus and multiple sclerosis; cellproliferative diseases, such as cancer, psoriasis and restenosis; andaccelerated muscle protein breakdown that accompanies variousphysiological and pathological states and is responsible to a largeextent for the loss of muscle mass (atrophy) that follows nerve injury,fasting, fever, acidosis, and certain endocrinopathies.

Compounds of the present invention inhibit the growth of cancer cells.Thus, the compounds can be employed to treat cancer, psoriasis,restenosis or other cell proliferative diseases in a patient in needthereof.

By the term “treatment of cancer” or “treating cancer” is intendeddescription of an activity of compounds of the present invention whereinsaid activity prevents or alleviates or ameliorates any of the specificphenomena known in the art to be associated with the pathology commonlyknown as “cancer.” The term “cancer” refers to the spectrum ofpathological symptoms associated with the initiation or progression, aswell as metastasis, of malignant tumors. By the term “tumor” isintended, for the purpose of the present invention, a new growth oftissue in which the multiplication of cells is uncontrolled andprogressive. The tumor that is particularly relevant to the invention isthe malignant tumor, one in which the primary tumor has the propertiesof invasion or metastasis or which shows a greater degree of anaplasiathan do benign tumors.

Thus, “treatment of cancer” or “treating cancer” refers to an activitythat prevents, alleviates or ameliorates any of the primary phenomena(initiation, progression, metastasis) or secondary symptoms associatedwith the disease. Cancers that are treatable are broadly divided intothe categories of carcinoma, lymphoma and sarcoma. Examples ofcarcinomas that can be treated by the composition of the presentinvention include, but are not limited to: adenocarcinoma, acinic celladeno carcinoma, adrenal cortical carcinomas, alveoli cell carcinoma,anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, renaladinol carcinoma,embryonal carcinoma, anometroid carcinoma, fibrolamolar liver cellcarcinoma, follicular carcinomas, giant cell carcinomas, hepatocellularcarcinoma, intraepidermal carcinoma, intraepithelial carcinoma,leptomanigio carcinoma, medullary carcinoma, melanotic carcinoma,menigual carcinoma, mesometonephric carcinoma, oat cell carcinoma,squamal cell carcinoma, sweat gland carcinoma, transitional cellcarcinoma, and tubular cell carcinoma. Sarcomas that can be treated bythe composition of the present invention include, but are not limitedto: amelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma,endometrial stroma sarcoma, ewing sarcoma, fascicular sarcoma, giantcell sarcoma, granulositic sarcoma, immunoblastic sarcoma, juxaccordialosteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (leukemia),lymphatic sarcoma (lympho sarcoma), medullary sarcoma, myeloid sarcoma(granulocitic sarcoma), austiogenci sarcoma, periostea sarcoma,reticulum cell sarcoma (histiocytic lymphoma), round cell sarcoma,spindle cell sarcoma, synovial sarcoma, and telangiectatic audiogenicsarcoma.

Lymphomas that can be treated by the composition of the presentinvention include, but are not limited to: Hodgkin's disease andlymphocytic lymphomas, such as Burkitt's lymphoma, NPDL, NML, NH anddiffuse lymphomas.

In other embodiments, certain of the proteasome pro-inhibitors employedin the practice of the present invention are capable of preventing thisactivation of NF-kB.

Blocking NF-kB activity is contemplated as possessing importantpractical application in various areas of medicine, e.g., inflammation,sepsis, AIDS, and the like.

In certain embodiments, the compounds of the present invention can beformulated in topical form for treatment of skin disorders selected frompsoriasis, dermatitis, Lichen planus, acne, and disorders marked byhyperproliferation of skin cells.

In certain embodiments, the compounds of the present invention can beformulated in topical form for treatment of uncontrolled hair growth.

(viii). Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, in certain cases it is preferable to administer thecompound as a pharmaceutical formulation (composition). Proteaseinhibitors according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine. In certain embodiments, the compound included in thepharmaceutical preparation may be active itself, or may be a prodrug,e.g., capable of being converted to an active compound in aphysiological setting.

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and, ifdesired, the active ingredient may be mixed with any conventionaladditive, such as an excipient, a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, or a coating agent. Although the dosage will varydepending on the symptoms, age and body weight of the patient, thenature and severity of the disorder to be treated or prevented, theroute of administration and the form of the drug, in general, a dailydosage of from 0.01 to 2000 mg of the compound is recommended for anadult human patient, and this may be administered in a single dose or individed doses.

The precise time of administration and/or amount of the compound thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. In certain embodiments, pharmaceutical compositions of thepresent invention are non-pyrogenic, i.e., do not induce significanttemperature elevations when administered to a patient.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the compound(s).These salts can be prepared in situ during the final isolation andpurification of the compound(s), or by separately reacting a purifiedcompound(s) in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts, and the like. (See, e.g., Berge et al. J. Pharm.Sci. 1977, 66, 1-19)

In other cases, the compounds useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an compound(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the compound(s), or by separately reacting the purified compound(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, e.g., Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol, and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated and the particular mode of administration. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of 100%,this amount will range from about 1% to about 99% of active ingredient,preferably from about 5% to about 70%, most preferably from about 10% toabout 30%.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound(s) with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a ligand with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of a compound(s) as an active ingredient. Acompound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose, and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets, and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compound(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more compound(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of acompound(s) include powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches, and inhalants. The active component may bemixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition tocompound(s), excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc, and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to a compound(s), excipientssuch as lactose, talc, silicic acid, aluminum hydroxide, calciumsilicates, and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The compound(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the compound. A non-aqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars, or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Medicaments which may be administered in inhalant or aerosolformulations according to the invention include protease inhibitorprodrugs useful in inhalation therapy which may be presented in a formwhich is soluble or substantially soluble in the selected propellantsystem.

The particle size of the particulate medicament should be such as topermit inhalation of substantially all of the medicament into the lungsupon administration of the aerosol formulation and will thus desirablybe less than 20 microns, preferably in the range 1 to 10 microns, e.g.,1 to 5 microns. The particle size of the medicament may be reduced byconventional means, for example by milling or micronisation.

Administration of medicament may be indicated for the treatment of mild,moderate or severe acute or chronic symptoms or for prophylactictreatment. It will be appreciated that the precise dose administeredwill depend on the age and condition of the patient, the particularparticulate medicament used and the frequency of administration and willultimately be at the discretion of the attendant physician. Whencombinations of medicaments are employed the dose of each component ofthe combination will in general be that employed for each component whenused alone. Typically, administration may be one or more times, forexample from 1 to 8 times per day, giving for example 1, 2, 3 or 4 puffseach time. Preferably, administration may be one time per day.

For administration, the drug is suitably inhaled from a nebulizer, froma pressurized metered dose inhaler, or as a dry powder from a dry powderinhaler (e.g., sold as TURBUHALER®) or from a dry powder inhalerutilizing gelatin, plastic or other capsules, cartridges or blisterpacks.

A diluent or carrier, generally non-toxic and chemically inert to themedicament; e.g., lactose, dextran, mannitol, glucose or any additivesthat will give the medicament a desired taste, can be added to thepowdered medicament.

The micronized mixture may be suspended or dissolved in a liquidpropellant mixture which is kept in a container that is sealed with ametering valve and fitted into a plastic actuator. The propellants usedmay be halocarbons of different chemical formulae. The most frequentlyused halocarbon propellants are trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethane, tetrafluoroethane,and 1,1-difluoroethane. Low concentrations of a surfactant such assorbitan trioleate, lecithin, disodium dioctylsulphosuccinate, or oleicacid may also be used to improve the physical stability.

Transdermal patches have the added advantage of providing controlleddelivery of a compound(s) to the body. Such dosage forms can be made bydissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the peptidomimeticin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions, and thelike, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds(s) in combination with oneor more pharmaceutically acceptable sterile isotonic aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofcompound(s) in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue.

When the compounds(s) of the present invention are administered aspharmaceuticals to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are of course given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as Applied Animal Nutrition; SanFrancisco: Freedman, 1969; or Livestock Feeds and Feeding; Corvallis: 0& B Books, 1977).

Regardless of the route of administration selected, the compound(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

(ix). Pharmaceutical Packages and Manufacture.

One aspect of the present invention provides a packaged pharmaceuticalcomprising one or more inhibitors of the present invention formulated ina pharmaceutically acceptable excipient, in association withinstructions (written and/or pictorial) describing the recommendeddosage and/or administration of the formulation to a patient. Suchinstructions may include details for treating or preventing a diseases,and optionally, warnings of possible side effects and drug-drug ordrug-food interactions.

Another aspect of the invention relates to the use of the subjectinhibitors in the manufacture of a medicament for the treatment of adisorder for which inhibition of the target protease of the inhibitormoiety G provides a therapeutic benefit to a patient. Exemplarydisorders are enumerated below.

Yet another aspect of the invention relates to a method for conducting apharmaceutical business, which includes:

a. manufacturing one or more of the subject inhibitors; and

b. marketing to healthcare providers the benefits of using thepreparation to treat or prevent any of the diseases or indications citedherein.

In certain embodiments, the subject business method can includeproviding a distribution network for selling the preparation. It mayalso include providing instruction material to patients or physiciansfor using the preparation to treat and prevent any of the diseases orindications cited herein.

(x). Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g., a variegated library of compoundsrepresented above, can be screened rapidly in high throughput assays inorder to identify potential protease inhibitor lead compounds, as wellas to refine the specificity, toxicity, and/or cytotoxic-kinetic profileof a lead compound.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectprotease inhibitors. See, for example, Blondelle et al. Trends Anal.Chem. 1995, 14, 83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al.J. Am. Chem. Soc. 1994, 116, 2661: Kerr et al. J. Am. Chem. Soc. 1993,115, 252; PCT publications WO92/10092, WO93/09668 and WO91/07087; andthe Lerner et al. PCT publication WO93/20242). Accordingly, a variety oflibraries on the order of about 100 to 1,000,000 or more diversomers ofthe subject protease inhibitors can be synthesized and screened forparticular activity or property.

In an exemplary embodiment, a library of candidate protease inhibitordiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolysable or photolyzablegroup, optionally located at one of the positions of the candidateagonists or a substituent of a synthetic intermediate. According to theStill et al. technique, the library is synthesized on a set of beads,each bead including a set of tags identifying the particular diversomeron that bead. The bead library can then be “plated” with proteases forwhich an inhibitor is sought. The diversomers can be released from thebead, e.g., by hydrolysis.

The structures of the compounds useful in the present invention lendthemselves readily to efficient synthesis. The nature of the structuresof the subject compounds, as generally set forth above, allows the rapidcombinatorial assembly of such compounds. For example, as in the schemeset forth below, an activated aryl group, such as an aryl triflate orbromide, attached to a bead or other solid support can be linked toanother aryl group by performing a Stille or Suzuki coupling with anaryl stannane or an aryl boronic acid. If the second aryl group isfunctionalized with an aldehyde, an amine substituent can be addedthrough a reductive amination. Alternatively, the second aryl groupcould be functionalized with a leaving group, such as a triflate,tosylate, or halide, capable of being displaced by an amine. Or, thesecond aryl group may be functionalized with an amine group capable ofundergoing reductive amination with an amine, e.g., CyKNH₂. Otherpossible coupling techniques include transition metal-mediated aminearylation reactions. The resultant secondary amine can then be furtherfunctionalized by an acylation, alkylation, or arylation to generate atertiary amine or amide which can then be cleaved from the resin orsupport. These reactions generally are quite mild and have beensuccessfully applied in combinatorial solid-phase synthesis schemes.Furthermore, the wide range of substrates and coupling partners suitableand available for these reactions permits the rapid assembly of large,diverse libraries of compounds for testing in assays as set forthherein. For certain schemes, and for certain substitutions on thevarious substituents of the subject compounds, one of skill in the artwill recognize the need for masking certain functional groups with asuitable protecting group. Such techniques are well known in the art andare easily applied to combinatorial synthesis schemes.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds which may be tested as proteaseinhibitors.

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill without departingfrom the spirit and the scope of the invention. Accordingly, theinvention is not to be limited only to the preceding illustrativedescription. For additional illustrative features that may be used withthe invention, including the embodiments described here, refer to thedocuments listed herein above and incorporated by reference in theirentirety. All operative combinations between the above describedillustrative embodiments and those features described below areconsidered to be potentially patentable embodiments of the invention.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 General Procedure for Synthesis of Thioxo-Amide-ContainingDipeptide (boro)Amino-Acid-Containing Analogues (Xaa-(C(S))-(boro)Xaa′Overview

Initially, a B-terminal protected form of a (boro)amino acid analogue isacylated with an activated form of an N-protected amino acid (oroligopeptide) to give a B-protected and N-protected (boro)aminoacid-containing dipeptide (or oligopeptide) analogue. The B-protectedand N-protected (boro)amino acid analogue is then transformed to thecorresponding thioxo amide compound using, e.g., Lawesson's Reagent.Finally, the thioxo-amide-containing (boro)amino acid dipeptide (oroligopeptide) analogue is deprotected to provide thethioxo-amide-containing (boro)amino acid dipeptide (or oligopeptide).

Application to Preparation of Ala-boroPro (Thioxo Amide)

To a stirred solution of N-Boc-L-Alanine (1.9 g, 10 mmol) andL-boroPro(pn). HCl (2.9 g, 10 mmol) in anhydrous DMF (30 mL) were addedHATU (4.0 mg, 10.5 mmol) and N,N-diisopropylethylamine (DIPEA, 4.0 mL,23 mmol) at 0° C. under argon atmosphere. The cooling bath was removedand the resulting mixture was stirred at room temperature for 1 hr, andthen was concentrated in vacuo under 30° C. The residue was dissolved inethyl acetate (100 mL), washed sequentially with KHSO₄ (0.1 M, 3×15 mL),aq. NaHCO₃ (5%, 3×10 mL), brine (3×10 mL) and dried (MgSO4) andevaporated. The crude product was purified by flash columnchromatography over silica gel (1:1, hexanes/EtOAc) to afford the purecoupling product as a white powder which was added to a stirredsuspension of Lawesson reagent (1.8 g, 4.5 mmol) in anhydrous toluene(100 mL) at room temperature. The resulting mixture was then stirred at80° C. for 4 hr. After removal of the solvent, the crude product waspurified by flash column chromatography over silica gel (2:1,hexanes/EtOAc) to afford N-Boc-Ala-boropro(pn) (thioxo amide) as a whitepowder. 3.1 g of this thioxo amide (7.1 mmol) was dissolved in anhydrousdichloromethane (40 mL), cooled to −78° C., a solution of borontrichloride in dichloromethane (35 mL, 1.0 M) was added and stirred for1 hr. The resulting mixture was evaporated to dryness under reducedpressure and co-evaporated using anhydrous methanol (3×15 mL). Theresidue was then partitioned between water (30 mL) and ether (30 mL).The aqueous phase was separated and washed with ether (2×20 mL).Concentrated the aqueous phase in vacuo and then purified bysemi-preparative RP-HPLC, lyophilized to afford the target compoundAla-boroPro (thioxo amide) as a white powder. ¹H NMR (D₂O, pH 2.01, δ):1.51 (d, J=6.7 Hz, 3H, CH₃), 1.83-1.90 (m, 1H, —CH₂CH_(A)H_(B)CHB—),2.08-2.25 (m, 3H, —CH₂CH_(A)H_(B)CHB—), 3.50-3.56 (m, 1H, —CH₂CH₂CHB—),3.62-3.69 (m, 1H, —NCH_(A)H_(B) CH₂CH₂—), 3.92-4.00 (m, 1H,—NCH_(A)H_(B)CH₂CH₂—), 4.55 (q, J=6.6 Hz, 1H, CH₃CHNH₂—); ¹¹B NMR (D₂O,pH 2.01, δ): 9.5; LC-MS (ESI+) for C₇H₁₅BN₂O₂S m/z (rel intensity):369.2 ([2×(M−H₂O)+H]⁺, 21), 203.1 ([M+H]⁺, 58), 186.1 ([M−NH₂]⁺, 100).HRMS: calcd for C₇H₁₆BN₂O₂S, [M+H]⁺, 203.1026, found 203.1030.

Example 2 DPIV Inhibition Assay

The inhibitor solution is prepared by dissolving 3-5 mg of inhibitor inpH 2 solution (0.01 N HCl), such that the concentration of the solutionis equal to 1 mg/10 mL. A 10 μL sample of this solution is then added to990 μL of pH 8 buffer (0.1 M HEPES, 0.14 M NaCl), and the solution isallowed to stand at room temperature overnight.

The enzyme solution is prepared by diluting 20 μL of DPIV (concentration2.5 nM) into 40 mL of pH 8 buffer.

The substrate solution is prepared by dissolving 2.0 mg ofL-alanyl-L-proline-para-nitroanilide into 20 mL of pH 8 buffer.

250 μL of enzyme solution is added to well #B1 to #H1, #A2 to #H2, and#A3 to #H3 of a 96 well plate, while well #A1 receives 250 μL of pH 8buffer instead of enzyme solution. 904 of pH 8 buffer is then added tocolumn 5 (from well #A5 to #H5).

A 1:10 dilution is then performed by adding inhibitor solution to #A5and the solution is mixed well before transferring 10 μL of thissolution from #A5 to #B5. The solution in #B5 is then mixed well beforetransferring 10 μL of this solution from #B5 to #C5. The solution in #C5is then mixed well before transferring 10 μL of this solution from #C5to #D5. The solution in #D5 is then mixed well before transferring 10 μLof this solution from #D5 to #E5. The solution in #E5 is then mixed wellbefore transferring 10 μL of this solution from #E5 to #F5. The solutionin #F5 is then mixed well before transferring 10 μL of this solutionfrom #F5 to #G5. The solution in #G5 is then mixed well beforetransferring 10 μL of this solution from #G5 to #H5.

A 30 μL aliquot is then transferred from #H5 to #H3 for row H, and thecontents are mixed well. The analogous procedure is repeated for rows G,F, E, D, C, B, and A sequentially. The plate is then shaken on a plateshaker for 5 min before allowing the plate to incubate at roomtemperature for an additional 5 min.

Once the plate has been allowed to incubate, 30 μL of substrate is addedto each well except well #A1. The plate is then placed on a plate shakerfor 5 min before allowing the plate to incubate at room temperature for25 min. The absorbance is then immediately read at a wavelength of 410nm.

Example 3 Selectivity for Dipeptidyl Peptidase Isoforms

The assay described in Example 2 is used to determine the IC₅₀ valuesfor several compounds of the invention. In this example, the assay isconducted for DPIV and DP8 or DP9. The ratio of IC₅₀ values for eachtested compound is calculated in order to determine the selectivity forthe DPIV isoform. IC₅₀ values were measured at the same pH throughoutthe assay.

Preferred compounds of the invention inhibit DPIV at least 10 times,preferably at least 100 times, more strongly than they inhibit DP8and/or DP9, i.e., have an IC₅₀ at least 10 (or 100) times lower againstDPIV than against DP8 and/or DP9.

Ratio Ratio DPP8 DP9 IC50s DPPIV DP8 DP9 to DPIV To DPIV Inhibitor pH 2pH 7.8 Ratio pH 2 pH 7.8 Ratio pH 2 pH 7.8 Ratio (pH 2) (pH 2) Pro-boro0.75 nM nd 2.5 nM 1.8 μM 720 1.9 nM 340 nM 180 3.2 2.5 Pro (thioxam)Pro-boro  76 nM 310 nM 4.1 140 nM  310 nM 2.2  85 nM 110 nM 1.3 1.8 1.1Ala (thioxam) Glu-boro  4.6 nM 890 nM 190  16 nM 2.0 μM 130 8.7 nM 1.8μM 180 3.4 1.9 Pro (thioxam) Ala-boro 0.74 nM 1.3 μM 1700 3.8 nM 2.4 μM630 8.4 nM 4.2 μM 500 5 11 Pro (thioxam)

Example 4 DPP IV Inhibition: Thioxamide v. Oxoamide

The inhibitory activity of a compound may be tested easily. DP-IV ispurified from pig kidney cortex by the method of Barth et al. (ActaBiol. Med. Germ. 32:157, 1974) and Wolf et al. (Acta Biol. Med. Germ.37:409, 1978) and from human placenta by the method of Puschel et al.(Eur. J. Biochem. 126:359, 1982). A compound is then screened for itsability to inhibit the protease activity of DP-IV with respect to anatural substrate. For example, the activity of DP-IV, isolated fromporcine kidneys by the method of Wolf et al. (ACTA Bio. Mes. Germ.37:409, 1972), was measured using Ala-Pro-p-nitroanilide as a substrate.Briefly, a reaction containing 50 micromol sodium Hepes (pH 7.8), 10micromol Ala-Pro-p-nitroanilide, 6 milliunits of DP-IV, and 2% (vol/vol)dimethylformamide in a total volume of 1.0 mL. The reaction wasinitiated by the addition of enzyme and reaction rates were measured at25 C; formation of reaction product (para-nitroanilide) in the presenceand absence of a test compound can be detected photometrically, e.g., at410 nm.

IC₅₀ IC₅₀ Inactivation Inhibitor (pH 2.0) (pH 8.0) Index Ala-boroPro

0.26 nM  1.4 uM  5,400 Ala-boroPro Thioxamide

0.35 nM  20 uM 57,000 Val-boroAla

 3.5 nM  9.0 nM    2.6 Val-boroAla Thioxamide

  73 nM 220 nM    3.0 Ala-boroAla

  63 nM 440 nM    7.0 Ala-boroAla Thioxamide

 6.5 uM  7.7 uM    1.2 Val-boroPro

 1.7 nM  1.2 uM   710 Val-boroPro Thioxamide

  44 nm  5.9 uM   130 Chg-boroPro

  1 nM 380 nM   380 Chg-boroPro Thioxamide

  4 nM  9 uM  2,300 Gly-boroPro

  1 nM  7 uM  7,000 Gly-boroPro Thioxamide

  3 nM  1.9 uM   630 NVP-LAF237 analogue

  42 nM  72 nM    1.7 NVP-LAF237 analogue Thioxamide

 160 nM  40 uM   250 N-(Benzyl)- Gly-boroPro

 9.5 nM  69 uM  7,300 N-(Benzyl)- Gly-boroPro Thioxamide

  24 nM 170 nM    7.1 Asp-boroPro

 200 nM  35 uM   180 Asp-boroPro Thioxamide

  17 nM  28 uM  1,700 Glu-boroPro

  6 nM  3 uM   500 Glu-boroPro Thioxamide

 4.6 nM 890 nM   190 Aad-boroPro

 4.5 nM  7.4 uM  1,600 Aad-boroPro Thioxamide

 1.6 nM  19 uM 12,000 Trp-boroPro

  2 nM  3 uM   1500 Trp-boroPro Thioxamide

 1.6 nM 700 nM   440 Arg-boroPro

 1.8 nM  7.1 uM  3,900 Arg-boroPro Thioxamide

 1.2 nM No inhibition N/A Pro-boroPro

 1.1 nM  23 uM 21,000 Pro-boroPro Thioxamide

 6.4 nM  7 uM  1,100

Example 5 X-BoroLeu Proteasome Inhibition: Thioxamide v. Oxoamide

The 26S proteasome is the multi-catalytic protease responsible for themajority of intracellular protein turnover in eukaryotic cells,including proteolytic degradation of damaged, oxidized or misfoldedproteins, as well as processing or degradation of key regulatoryproteins required for various cellular function (Ciechanover, Cell 79:13-21 (1994); Coux et al., Ann. Rev. Biochem. 65:801-847 (1995);Goldberg et al., Chemistry & Biology 2:503-508 (1995)). Proteinsubstrates are first marked for degradation by covalent conjugation tomultiple molecules of a small protein, ubiquitin. The resultantpolyubiquitinated protein is then recognized and degraded by the 26Sproteasome.

Constituting the catalytic core of the 26S proteasome is the 20Sproteasome, a multi-subunit complex of approximately 700 kDa molecularweight. Coux et al. (Ann. Rev. Biochem. 65:801-847 (1995)) teaches thatthe 20S proteasome does not by itself degrade ubiquitinated proteins,but does possess multiple peptidase activities. Based on substratepreferences, Coux et al. characterizes these activities aschymotrypsin-like, trypsin-like, post-glutamyl hydrolase, branched chainamino acid preferring, and small neutral amino acid preferring. Coux etal. also teaches that a dramatic activation of 20S proteasome activitycan be induced by various in vitro treatments, such as heating to55.degree. C., incubation with basic polypeptides, sodium dodecylsulfate (SDS), guanidine HCl or fatty acids, dialysis against water, orby physiological regulators such as PA28 or PA700. McCormack et al.(Biochemistry 37:7792-7800 (1998)) teaches that a variety of peptidesubstrates, including Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Arg-AMC, andZ-Leu-Leu-Glu-2NA, wherein Suc is N-succinyl, AMC is7-amino-4-methylcoumarin, and 2NA is 2-naphthylamine, are cleaved by the20S proteasome.

The ubiquitin-proteasome pathway plays a central role in a large numberof physiological processes. Deshaies (Trends in Cell Biol. 5: 428-434(1995)) and Hoyt (Cell 91:149-151 (1997)) teach that regulatedproteolysis of cell cycle proteins, including cyclins, cyclin-dependentkinase inhibitors, and tumor suppressor proteins, is required forcontrolled cell cycle progression and that proteolysis of these proteinsoccurs via the ubiquitin-proteasome pathway. Palombella et al., WO95/25533 teaches that activation of the transcription factor NF-kappa-B,which itself plays a central role in the regulation of genes involved inthe immune and inflammatory responses, is dependent upon theproteasome-mediated degradation of an inhibitory protein,Ikappa-B-.alpha. Goldberg and Rock, WO 94/17816 discloses that thecontinual turnover of cellular proteins by the ubiquitin-proteasomepathway plays an essential role in antigen presentation.

Inhibition of proteasome activity thus offers a promising new approachfor therapeutic intervention in these and other conditions directly orindirectly mediated by the proteolytic function of the proteasome.Goldberg et al. (Chemistry & Biology 2:503-508 (1995)) teaches thatproteasome inhibitors block the inflammatory response in vivo in animalmodels of human disease.

Compounds may be screened for their ability to inhibit theATP-ubiquitin-dependent degradative process by measurement proteolysisin cultured cells (Rock et al., Cell, vol. 78:761 (1994)). For example,the degradation of long-lived intracellular proteins can be measured inmouse C2C12 myoblast cells. Cells are incubated with ³⁵S-methionine for48 hours to label long-lived proteins and then chased for 2 hours withmedium containing unlabeled methionine. After the chase period, thecells are incubated for 4 hours in the presence or absence of the testcompound. The amount of protein degradation in the cell can be measuredby quantitating the trichloroacetic acid soluble radioactivity releasedfrom the prelabeled proteins into the growth medium (an indicator ofintracellular proteolysis).

Compounds can also be tested for their ability to reduce muscle wastingin vivo. Urinary excretion of the modified amino acid 3-methyl histidine(3-MH) is probably the most well characterized method for studyingmyofibrillar protein degradation in vivo (see Young and Munro,Federation Proc. 37:229-2300 (1978)). 3-Methylhistidine is apost-translationally modified amino acid which cannot be reutilized forprotein synthesis, and it is only known to occur in actin and myosin. Itoccurs in actin isolated from all sources, including cytoplasmic actinfrom many different cell types. It also occurs in the myosin heavy chainof fast-twitch (white, type II) muscle fibers, but it is absent frommyosin of cardiac muscle and myosin of slow-twitch (red, type I) musclefibers. Due to its presence in actin of other tissues than skeletalmuscle, other tissues will contribute to urinary 3-MH. Skeletal musclehas been estimated to contribute 38-74% of the urinary 3-MH in normalrats and 79-86% of the urinary 3-MH in rats treated with corticosterone(100 mg/kg/day subcutaneously) for 2-4 days (Millward and Bates,Biochem. J. 214:607-615 (1983); Kayali, et al., Am. J. Physiol.252:E621-E626 (1987)).

High-dose glucocorticoid treatment can be used to induce a state ofmuscle wasting in rats. Treating rats with daily subcutaneous injectionsof corticosterone (100 mg/kg) causes an increase of approximately 2-foldin urinary 3-MH. The increase in excretion of 3-MH is transient, with apeak increase after 2-4 days of treatment and a return to basal valuesafter 6-7 days of treatment (Odedra, et al., Biochem. J. 214:617-627(1983); Kayali, et al., Am. J. Physiol. 252:E621-E626 (1987)).Glucocorticoids have been shown to activate the ATP-ubiquitin-dependentproteolytic pathway in skeletal muscle (Wing and Goldberg, Am. J.Physiol. 264:E668-E676 (1993)) and proteasome inhibitors are thereforeexpected to inhibit the muscle wasting that occurs after glucocorticoidtreatment.

IC₅₀ IC₅₀ Inactivation Inhibitor (ph 2.0) (ph 7.6) Index Ala-boroLeu

  31 nM 100 nM 3.2 Ala-boroLeu Thioxamide

0.85 uM  92 uM 110 Asp-boroLeu Thioxamide

 4.6 uM  10 uM 2.2 Phe-boroLeu (free Velcade)

  16 nM 120 nM 7.5 Phe-boroLeu Thioxamide

0.71 uM  14 uM 20 Gly-boroLeu

  14 nM 160 nM 11 Gly-boroLeu Thioxamide

 4.9 uM 100 uM 20 Pyz-Gly- boroLeu

 100 nM 120 nM 1.2 N-(Pyrazine-2- carbothio)- Gly-boroLeu

  77 nM 350 nM 4.5 N-(Pyrazine-2- carbonyl)-Gly- boroLeu Thioxamide

 5.8 uM  22 uM 3.8 Pyz-Gly- boroLeu Perthioxamide

 3.9 uM  2.4 mM 620

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for inhibiting the proteolytic activity of apost-proline-cleaving enzyme, wherein said enzyme is a mammaliandipeptidyl peptidase IV (DPP IV), comprising contacting said enzyme witha compound represented by:

or a pharmaceutically acceptable salt thereof; wherein the bond betweenR₁ and N is a thioxamide bond; A represents a 4-8 membered heterocycleincluding the N and the Cα carbon; R₁ represents a C-terminally linkedamino acid residue or amino acid analog, or a C-terminally linkedpeptide or peptide analog; or

R₂ is absent or represents one or more substitutions to the ring A, eachof which is independently a halogen, lower alkyl, lower alkenyl, loweralkynyl, carbonyl, carboxyl, ester, formate, ketone, thiocarbonyl,thioester, thioacetate, thioformate, amino, acylamino, amido, nitro,sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato,

R₃ represents hydrogen or a halogen, lower alkyl, lower alkenyl, loweralkynyl, carbonyl, thiocarbonyl, amino, acylamino, amido, nitro,sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)—,O-lower alkyl, —(CH₂)—, —O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)S—(CH₂)_(m)—R₇, azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

R₇, independently for each occurrence, represents a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;R₈, independently for each occurrence, represents hydrogen, —CH₃, or—(CH₂)_(n)—CH₃; Y₁ and Y₂, independently, are OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, or Y₁ and Y₂ are connected via aring having from 5 to 8 atoms in the ring structure which is capable ofbeing hydrolyzed to two hydroxyl groups; m is zero or an integer in therange of 1 to 8; and n is an integer in the range of 1 to
 8. 2-64.(canceled)
 65. The method of claim 1, wherein Y₁ and Y₂, independently,are OH.
 66. The method of claim 1, wherein said compound is representedby:


67. The method of claim 1, wherein R₂ is absent, or represents loweralkyl or halogen.
 68. The method of claim 1, wherein R₃ is hydrogen. 69.The method of claim 1, wherein the Cα carbon exists substantially in theR configuration.
 70. The method of claim 1, wherein the Cα carbon existssubstantially in the S configuration.
 71. The method of claim 1, whereinthe Cα carbon exists in a racemic mixture of R and S configurations. 72.The method of claim 1, wherein R₁ is a proline, glutamate, or alanineresidue.
 73. The method of claim 1, wherein R₁ is an alanine residue.74. The method of claim 1, wherein selected from the group consistingof:

and enantiomers, diastereomers, and salts thereof.
 75. The method ofclaim 1, wherein the compound inhibits dipeptidyl peptidase IV with a Kiof 50 nanomolar (nM) or less.
 76. The method of claim 75, wherein thecompound inhibits dipeptidyl peptidase VIII and IX with a Ki of 100micromolar (μM) or greater.
 77. A method of regulating glucosemetabolism in a patient, comprising administering to a patient in needthereof a therapeutically effective amount of a compound represented by:

or a pharmaceutically acceptable salt thereof; wherein the bond betweenR₁ and N is a thioxamide bond; A represents a 4-8 membered heterocycleincluding the N and the Cα carbon; R₁ represents a C-terminally linkedamino acid residue or amino acid analog, or a C-terminally linkedpeptide or peptide analog; R₂ is absent or represents one or moresubstitutions to the ring A, each of which is independently a halogen,lower alkyl, lower alkenyl, lower alkynyl, carbonyl, carboxyl, ester,formate, ketone, thiocarbonyl, thioester, thioacetate, thioformate,amino, acylamino, amido, nitro, sulfate, sulfonate, sulfonamido,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇, azido, cyano, isocyanato, thiocyanato,isothiocyanato, cyanato,

R₃ represents hydrogen or a halogen, lower alkyl, lower alkenyl, loweralkynyl, carbonyl, thiocarbonyl, amino, acylamino, amido, nitro,sulfate, sulfonate, sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇, azido, cyano,isocyanato, thiocyanato, isothiocyanato, cyanato

R₇, independently for each occurrence, represents a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;R₈, independently for each occurrence, represents hydrogen, —CH₃, or—(CH₂)_(n)—CH₃; Y₁ and Y₂, independently, are OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, or Y₁ and Y₂ are connected via aring having from 5 to 8 atoms in the ring structure which is capable ofbeing hydrolyzed to two hydroxyl groups; m is zero or an integer in therange of 1 to 8; and n is an integer in the range of 1 to
 8. 78. Themethod of claim 77, wherein Y₁ and Y₂, independently, are OH.
 79. Themethod of claim 77, wherein said compound is represented by:


80. The method of claim 77, wherein R₂ is absent, or represents loweralkyl or halogen.
 81. The method of claim 77, wherein R₃ is hydrogen.82. The method of claim 77, wherein the Cα carbon exists substantiallyin the R configuration.
 83. The method of claim 77, wherein the Cαcarbon exists substantially in the S configuration.
 84. The method ofclaim 77, wherein the Cα carbon exists in a racemic mixture of R and Sconfigurations.
 85. The method of claim 77, wherein R₁ is a proline,glutamate, or alanine residue.
 86. The method of claim 77, wherein R₁ isan alanine residue.
 87. The method of claim 77, wherein selected fromthe group consisting of:

and enantiomers, diastereomers and salts thereof.